Methods for treating hepatocellular cancer by administering an anti-Ang-2 antibody

ABSTRACT

The present invention provides antibodies that bind to angiopoietin-2 (Ang-2) and methods of using same. According to certain embodiments of the invention, the antibodies are fully human antibodies that bind to human Ang-2. The antibodies of the invention are useful, inter alia, for the treatment of diseases and disorders associated with one or more Ang-2 biological activities including angiogenesis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. non-provisional application Ser. No. 13/747,728, filed Jan. 23, 2013, which is a continuation-in-part of U.S. non-provisional application Ser. No. 12/843,905, filed on Jul. 27, 2010, which claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. provisional application No. 61/229,418, filed on Jul. 29, 2009, and U.S. provisional application No. 61/295,194, filed on Jan. 15, 2010, the disclosures of which are herein incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to antibodies, and antigen-binding fragments thereof, which are specific for angiopoietin-2 (Ang-2).

SEQUENCE LISTING

This application includes an electronic sequence listing in a file named “457434-Sequence.txt”, created on Feb. 6, 2015 and containing 304,998 bytes, which is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND

Angiogenesis is the biological process whereby new blood vessels are formed. Aberrant angiogenesis is associated with several disease conditions including, e.g., proliferative retinopathies, rheumatoid arthritis and psoriasis. In addition, it is well established that angiogenesis is critical for tumor growth and maintenance. Angiopoietin-2 (Ang-2) is a ligand for the Tie-2 receptor (Tie-2) and has been shown to play a role in angiogenesis. Ang-2 is also referred to in the art as Tie-2 ligand. (U.S. Pat. No. 5,643,755; Yancopoulos et al., 2000, Nature 407:242-248).

Antibodies and other peptide inhibitors that bind to Ang-2 are mentioned in, e.g., U.S. Pat. Nos. 6,166,185; 7,521,053; 7,205,275; 2006/0018909 and 2006/0246071. There is a need in the art for novel Ang-2 modulating agents, including Ang-2 antibodies, which can be used to treat diseases and conditions caused by or exacerbated by angiogenesis.

BRIEF SUMMARY OF THE INVENTION

The present invention provides human antibodies that bind to human Ang-2. The present inventors, in view of various lines of evidence and investigation, have recognized a need for Ang-2 inhibitors which do not bind to or antagonize the related molecule Ang-1. For example, previous studies have demonstrated or suggested a beneficial role for Ang-1 in hemostasis (see, e.g., Li et al., 2001, Thrombosis and Haemostasis 85:191-374) and in protecting the adult vasculature against plasma leakage (see, e.g., Thurston et al., 2000, Nature Medicine 6:460-463; Thurston et al., 1999, Science 286:2511-2514). Thus, the present inventors recognized that, in certain anti-angiogenic therapeutic situations, it may be beneficial to preserve Ang-1 activity. Accordingly, the present invention provides antibodies which bind specifically to Ang-2 but do not substantially bind to Ang-1. The present invention also includes antibodies that block the interaction between Ang-2 and its receptor Tie-2 but do not substantially block the interaction between Ang-1 and Tie-2. The antibodies of the invention are useful, inter alia, for inhibiting the angiogenesis-promoting activities of Ang-2 and for treating diseases and disorders caused by or related to the process of angiogenesis. The invention also provides for methods of treating cancer, the methods comprising administering therapeutically effective amounts of anti-Ang-2 antibodies, as described herein.

The antibodies of the invention can be full-length (for example, an IgG1 or IgG4 antibody) or may comprise only an antigen-binding portion (for example, a Fab, F(ab′)₂ or scFv fragment), and may be modified to affect functionality, e.g., to eliminate residual effector functions.

In one embodiment, the invention comprises an antibody or antigen-binding fragment of an antibody comprising a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 18, 22, 26, 42, 46, 50, 66, 70, 74, 90, 94, 98, 114, 118, 122, 138, 142, 146, 162, 166, 170, 186, 190, 194, 210, 214, 218, 234, 238, 242, 258, 262, 266, 282, 286, 290, 306, 310, 314, 330, 334, 338, 354, 358, 362, 378, 382, 386, 402, 406, 410, 426, 430, 434, 450, 454, 458, 474, 478, 482, 498, 502, 506, 514, and 516, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. In one embodiment, the antibody or antigen-binding portion of an antibody comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 42, 66, 162, 210, 266, and 434.

In one embodiment, the invention comprises an antibody or antigen-binding fragment of an antibody comprising a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 20, 24, 34, 44, 48, 58, 68, 72, 82, 92, 96, 106, 116, 120, 130, 140, 144, 154, 164, 168, 178, 188, 192, 202, 212, 216, 226, 236, 240, 250, 260, 264, 274, 284, 288, 298, 308, 312, 322, 332, 336, 346, 356, 360, 370, 380, 384, 394, 404, 408, 418, 428, 432, 442, 452, 456, 466, 476, 480, 490, 500, and 504, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. In one embodiment, the antibody or antigen-binding portion of an antibody comprises a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 20, 44, 68, 164, 212, 274, and 442.

In specific embodiments, the antibody or antigen-binding fragment thereof comprises a HCVR and LCVR (HCVR/LCVR) amino acid sequence pair selected from the group consisting of SEQ ID NO: 2/10, 18/20, 22/24, 26/34, 42/44, 46/48, 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 98/106, 114/116, 118/120, 122/130, 138/140, 142/144, 146/154, 162/164, 166/168, 170/178, 186/188, 190/192, 194/202, 210/212, 214/216, 218/226, 234/236, 238/240, 242/250, 258/260, 262/264, 266/274, 282/284, 286/288, 290/298, 306/308, 310/312, 314/322, 330/332, 334/336, 338/346, 354/356, 358/360, 362/370, 378/380, 382/384, 386/394, 402/404, 406/408, 410/418, 426/428, 430/432, 434/442, 450/452, 454/456, 458/466, 474/476, 478/480, 482/490, 498/500, and 502/504. In one embodiment, the antibody or fragment thereof comprises a HCVR and LCVR selected from the amino acid sequence pairs of SEQ ID NO: 18/20, 42/44, 66/68, 162/164, 210/212, 266/274, and 434/442.

In a next aspect, the invention provides an antibody or antigen-binding fragment of an antibody comprising a heavy chain CDR3 (HCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 32, 56, 80, 104, 128, 152, 176, 200, 224, 248, 272, 296, 320, 344, 368, 392, 416, 440, 464, 488, and 512, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a light chain CDR3 (LCDR3) domain selected from the group consisting of SEQ ID NO: 16, 40, 64, 88, 112, 136, 160, 184, 208, 232, 256, 280, 304, 328, 352, 376, 400, 424, 448, 472, and 496, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

In certain embodiments, the antibody or antigen-binding portion of an antibody comprises a HCDR3/LCDR3 amino acid sequence pair selected from the group consisting of SEQ ID NO: 8/16, 32/40, 56/64, 80/88, 104/112, 128/136, 152/160, 176/184, 200/208, 224/232, 248/256, 272/280, 296/304, 320/328, 344/352, 368/376, 392/400, 416/424, 440/448, 464/472, and 488/496. In one embodiment, the antibody or antigen-binding portion of an antibody comprises a HCDR3/LCDR3 amino acid sequence pair selected from the group consisting of SEQ ID NO: 8/16, 32/40, 56/64, 152/160, 200/208, 272/280, and 440/448. Non-limiting examples of anti-Ang-2 antibodies having these HCDR3/LCDR3 pairs are the antibodies designated H1H685, H1H690, H1H691, H1H696, H1H706, H1M724, and H2M744, respectively.

In a further embodiment, the invention comprises an antibody or fragment thereof further comprising a HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 28, 52, 76, 100, 124, 148, 172, 196, 220, 244, 268, 292, 316, 340, 364, 388, 412, 436, 460, 484, and 508, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a heavy chain CDR2 (HCDR2) domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 30, 54, 78, 102, 126, 150, 174, 198, 222, 246, 270, 294, 318, 342, 366, 390, 414, 438, 462, 486, and 510, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 36, 60, 84, 108, 132, 156, 180, 204, 228, 252, 276, 300, 324, 348, 372, 396, 420, 444, 468, and 492, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 38, 62, 86, 110, 134, 158, 182, 206, 230, 254, 278, 302, 326, 350, 374, 398, 422, 446, 470, and 494, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

Certain non-limiting, exemplary antibodies and antigen-binding fragments of the invention comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 domains, respectively, selected from the group consisting of: (i) SEQ ID NO: 4, 6, 8, 12, 14 and 16 (e.g., H1H685); (ii) SEQ ID NO: 28, 30, 32, 36, 38 and 40 (e.g., H1H690); (iii) SEQ ID NO: 52, 54, 56, 60, 62 and 64 (e.g., H1H691); (iv) SEQ ID NO: 148, 150, 152, 156, 158 and 160 (e.g., H1H696); (v) SEQ ID NO: 196, 198, 200, 204, 206 and 208 (e.g., H1H706); (vi) SEQ ID NO: 268, 270, 272, 276, 278 and 280 (e.g., H1M724); and (vii) SEQ ID NO: 436, 438, 440, 444, 446 and 448 (e.g., H2M744).

In a related embodiment, the invention comprises an antibody or antigen-binding fragment of an antibody which specifically binds Ang-2, wherein the antibody or fragment comprises the heavy and light chain CDR domains (i.e., CDR1, CDR2 and CDR3) contained within heavy and light chain variable domain sequences selected from the group consisting of SEQ ID NO: 2/10, 18/20, 22/24, 26/34, 42/44, 46/48, 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 98/106, 114/116, 118/120, 122/130, 138/140, 142/144, 146/154, 162/164, 166/168, 170/178, 186/188, 190/192, 194/202, 210/212, 214/216, 218/226, 234/236, 238/240, 242/250, 258/260, 262/264, 266/274, 282/284, 286/288, 290/298, 306/308, 310/312, 314/322, 330/332, 334/336, 338/346, 354/356, 358/360, 362/370, 378/380, 382/384, 386/394, 402/404, 406/408, 410/418, 426/428, 430/432, 434/442, 450/452, 454/456, 458/466, 474/476, 478/480, 482/490, 498/500, and 502/504. In one embodiment, the antibody or fragment thereof comprises the CDR sequences contained within HCVR and LCVR selected from the amino acid sequence pairs of SEQ ID NO: 18/20, 42/44, 66/68, 162/164, 210/212, 266/274, and 434/442.

In another aspect, the invention provides nucleic acid molecules encoding anti-Ang-2 antibodies or fragments thereof. Recombinant expression vectors carrying the nucleic acids of the invention, and host cells into which such vectors have been introduced, are also encompassed by the invention, as are methods of producing the antibodies by culturing the host cells under conditions permitting production of the antibodies, and recovering the antibodies produced.

In one embodiment, the invention provides an antibody or fragment thereof comprising a HCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 17, 21, 25, 41, 45, 49, 65, 69, 73, 89, 93, 97, 113, 117, 121, 137, 141, 145, 161, 165, 169, 185, 189, 193, 209, 213, 217, 233, 237, 241, 257, 261, 265, 281, 285, 289, 305, 309, 313, 329, 333, 337, 353, 357, 361, 377, 381, 385, 401, 405, 409, 425, 429, 433, 449, 453, 457, 473, 477, 481, 497, 501, 505, 513, and 515, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity thereto. In one embodiment, the antibody or fragment thereof comprises a HCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 17, 41, 65, 161, 209, 265, and 433.

In one embodiment, the invention provides an antibody or fragment thereof comprising a LCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, 19, 23, 33, 43, 47, 57, 67, 71, 81, 91, 95, 105, 115, 119, 129, 139, 143, 153, 163, 167, 177, 187, 191, 201, 211, 215, 225, 235, 239, 249, 259, 263, 273, 283, 287, 297, 307, 311, 321, 331, 335, 345, 355, 359, 369, 379, 383, 393, 403, 407, 417, 427, 431, 441, 451, 455, 465, 475, 479, 489, 499, and 503, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity thereto. In one embodiment, the antibody or fragment thereof comprises a LCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 19, 43, 67, 163, 211, 273, and 441.

In one embodiment, the invention provides an antibody or antigen-binding fragment of an antibody comprising a HCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 7, 31, 55, 79, 103, 127, 151, 175, 199, 223, 247, 271, 295, 319, 343, 367, 391, 415, 439, 463, 487, and 511, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; and a LCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 15, 39, 63, 87, 111, 135, 159, 183, 207, 231, 255, 279, 303, 327, 351, 375, 399, 423, 447, 471, and 495, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity thereto. In one embodiment, the antibody or fragment thereof comprises HCDR3 and LCDR3 sequences encoded by the nucleic acid sequence pairs selected from the group consisting of SEQ ID NO: 7/15, 31/39, 55/63, 151/159, 199/207, 271/279, and 439/447.

In a further embodiment, the antibody or fragment thereof further comprises: a HCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, 27, 51, 75, 99, 123, 147, 171, 195, 219, 243, 267, 291, 315, 339, 363, 387, 411, 435, 459, 483, and 507, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; a HCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 5, 29, 53, 77, 101, 125, 149, 173, 197, 221, 245, 269, 293, 317, 341, 365, 389, 413, 437, 461, 485, and 509, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; a LCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 11, 35, 59, 83, 107, 131, 155, 179, 203, 227, 251, 275, 299, 323, 347, 371, 395, 419, 443, 467, and 491, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; and a LCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 13, 37, 61, 85, 109, 133, 157, 181, 205, 229, 253, 277, 301, 325, 349, 373, 397, 421, 445, 469, and 493, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.

In one embodiment, the antibody or fragment thereof comprises the heavy and light chain CDR sequences encoded by the nucleic acid sequences of SEQ ID NO: 17 and 19; SEQ ID NO: 41 and 43; SEQ ID NO: 65 and 67; SEQ ID NO: 161 and 163; SEQ ID NO: 209 and 211; SEQ ID NO: 265 and 273; or SEQ ID NO: 433 and 441.

The invention encompasses anti-Ang-2 antibodies having a modified glycosylation pattern. In some applications, modification to remove undesirable glycosylation sites may be useful. For example, the present invention encompasses modified versions of any antibody set forth herein wherein the modified version lacks a fucose moiety present on the oligosaccharide chain, for example, to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modification of galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).

In another aspect, the invention provides a pharmaceutical composition comprising a recombinant human antibody or fragment thereof which specifically binds Ang-2 and a pharmaceutically acceptable carrier or diluent. In a related aspect, the invention features a composition which is a combination of an Ang-2 inhibitor and a second therapeutic agent. In one embodiment, the Ang-2 inhibitor is an antibody or fragment thereof. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an Ang-2 inhibitor. Exemplary agents that may be advantageously combined with an Ang-2 inhibitor include, without limitation, any agent that inhibits or reduces angiogenesis, other cancer therapeutic agents, anti-inflammatory agents, cytokine inhibitors, growth factor inhibitors, anti-hematopoietic factors, non-steroidal anti-inflammatory drugs (NSAIDs), antiviral agents, and antibiotics.

In another aspect, the invention provides methods for inhibiting Ang-2 activity using the anti-Ang-2 antibody or antigen-binding portion of the antibody of the invention, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an antibody or antigen-binding fragment of an antibody of the invention. The disorder treated is any disease or condition which is improved, ameliorated, inhibited or prevented by removal, inhibition or reduction of Ang-2 activity. Preferably, the anti-Ang-2 antibody or antibody fragment of the invention is useful to treat any disease or condition caused by, associated with, or perpetuated by the process of angiogenesis. In certain embodiments of the invention, the anti-Ang-2 antibodies or antigen-binding portions thereof are useful for the treatment of cancer. In the context of cancer therapies, the anti-Ang-2 antibodies of the invention or antigen-binding portions thereof can be administered alone or in combination with other anti-cancer therapeutic antibodies, chemotherapeutic agents and/or radiation therapy. In other embodiments of the present invention, the anti-Ang-2 antibodies or antigen-binding fragments thereof are useful for the treatment of one or more eye disorders, e.g., age-related macular degeneration, diabetic retinopathy, etc., and/or one or more inflammatory or infectious diseases.

In yet another aspect, the invention provides methods for treating advanced cancer or solid tumor malignancies, wherein the methods comprise administering a therapeutically effective amount of an antibody or antigen binding fragment that specifically binds Ang-2 but does not substantially bind Ang-1. In certain embodiments, methods of the invention are used in treatment of advanced cancer that is resistant to conventional anti-cancer therapy. In some embodiments, the methods further comprise administering a second therapeutic agent. The second therapeutic agent may be another anti-angiogenic agent. An example of an anti-angiogenic agent that may be used in combination with the anti-Ang-2 antibodies in the methods of treatment of the present invention is aflibercept, a vascular endothelial growth factor (VEGF)-inhibiting fusion protein.

Other embodiments will become apparent from a review of the ensuing detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an alignment of the last 88 C-terminal amino acids of human Ang-2 (residues 409 to 496 of SEQ ID NO:518) with the corresponding amino acid sequence of human Ang-1 (SEQ ID NO:531). Residues that differ between hAng-1 and hAng-2 are indicated by white text and black shading. Asterisks (*) indicate the amino acids of hAng-2 which were shown to interact with human Tie-2 by crystal structure analysis. See Barton et al., Nat. Struct. Mol. Biol. 13:524-532 (2006). Triangles (▴) indicate the Tie-2-interacting amino acid positions that differ between hAng-2 and hAng-1.

FIG. 2 (Panels A-C) depict the results of Western blots which illustrate the extent to which Ang-2 binding molecules inhibit, or fail to inhibit, Ang-1-induced Tie-2 phosphorylation.

FIG. 3 is a summary of the Ang-2FD-mFc point mutant binding experiment of Example 13, showing the amino acid changes which resulted in greater than a five-fold reduction in T½ of dissociation (depicted by solid circles ●) relative to wild-type for the various antibodies and peptibodies tested.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood that this invention is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term “about,” when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression “about 100” includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications and non-patent publications mentioned in this specification are incorporated herein by reference in their entireties.

Definitions

As used herein, the term “angiopoietin-2” or “Ang-2”, unless specified as being from a non-human species (e.g., “mouse Ang-2,” “monkey Ang-2,” etc.), refers to human Ang-2 or a biologically active fragment thereof (e.g., a fragment of the Ang-2 protein which is capable of inducing angiogenesis in vitro or in vivo). Human Ang-2 is encoded by the nucleic acid sequence shown in SEQ ID NO:517 and has the amino acid sequence of SEQ ID NO:518. The amino acid sequences of mouse and monkey Ang-2 proteins are available from the NCBI protein sequence database under Accession Nos. NP_031452 and BAE89705.1, respectively.

The term “angiopoietin-1” or “Ang-1”, unless specified as being from a non-human species (e.g., “mouse Ang-1,” “monkey Ang-1,” etc.), refers to human Ang-1 or a biologically active fragment thereof. Human Ang-1 has the amino acid sequence as set forth in the NCBI protein sequence database under Accession No. AAB50557. The term “Tie-2” (also referred to in the art as “TEK”) unless specified as being from a non-human species (e.g., “mouse Tie-2,” “monkey Tie-2,” etc.), refers to human Tie-2 or a biologically active fragment thereof. Human Tie-2 has the amino acid sequence as set forth in the NCBI protein sequence database under Accession No. AAA61130.

The term “antibody”, as used herein, is intended to refer to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V_(H)) and a heavy chain constant region. The heavy chain constant region comprises three domains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V_(L)) and a light chain constant region. The light chain constant region comprises one domain (C_(L)1). The V_(H) and V_(L) regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each V_(H) and V_(L) is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the anti-Ang-2 antibody (or antigen-binding portion thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

The term “antibody,” as used herein, also includes antigen-binding fragments of full antibody molecules. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab′)₂ fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR)). Other engineered molecules, such as diabodies, triabodies, tetrabodies and minibodies, are also encompassed within the expression “antigen-binding fragment,” as used herein.

An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a V_(H) domain associated with a V_(L) domain, the V_(H) and V_(L) domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) or V_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present invention include: (i) V_(H)-C_(H)1; (ii) V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v) V_(H)-C_(H)1-C_(H)2-C_(H)3, (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L); (viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi) V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3, (xiii) V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present invention may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric V_(H) or V_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using routine techniques available in the art.

The constant region of an antibody is important in the ability of an antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of an antibody may be selected on the basis of whether it is desirable for the antibody to mediate cytotoxicity.

The term “human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The term “recombinant human antibody”, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V_(H) and V_(L) regions of the recombinant antibodies are sequences that, while derived from and related to human germline V_(H) and V_(L) sequences, may not naturally exist within the human antibody germline repertoire in vivo.

Human antibodies can exist in two forms that are associated with hinge heterogeneity. In one form, an immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond. In a second form, the dimers are not linked via inter-chain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody). These forms have been extremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed using a human IgG1 hinge. The instant invention encompasses antibodies having one or more mutations in the hinge, C_(H)2 or C_(H)3 region which may be desirable, for example, in production, to improve the yield of the desired antibody form.

An “isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds human Ang-2 or a human Ang-2 fragment is substantially free of antibodies that specifically bind antigens other than human Ang-2). The term “specifically binds,” or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by a K_(D) of about 1×10⁻⁸ M or less. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. An isolated antibody that specifically binds human Ang-2 may, however, have cross-reactivity to other antigens, such as Ang-2 molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

A “neutralizing” or “blocking” antibody, as used herein, is intended to refer to an antibody whose binding to Ang-2 blocks the interaction between Ang-2 and its receptor (Tie-2) and/or results in inhibition of at least one biological function of Ang-2. The inhibition caused by an Ang-2 neutralizing or blocking antibody need not be complete so long as it is detectable using an appropriate assay. Exemplary assays for detecting Ang-2 inhibition are described elsewhere herein.

The fully-human anti-Ang-2 antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The present invention includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are back-mutated to the corresponding germline residue(s) or to a conservative amino acid substitution (natural or non-natural) of the corresponding germline residue(s) (such sequence changes are referred to herein as “germline back-mutations”). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline back-mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR residues within the V_(H) and/or V_(L) domains are mutated back to the germline sequence. In other embodiments, only certain residues are mutated back to the germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. Furthermore, the antibodies of the present invention may contain any combination of two or more germline back-mutations within the framework and/or CDR regions, i.e., wherein certain individual residues are mutated back to the germline sequence while certain other residues that differ from the germline sequence are maintained. Once obtained, antibodies and antigen-binding fragments that contain one or more germline back-mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present invention.

The present invention also includes anti-Ang-2 antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes anti-Ang-2 antibodies having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein. In one embodiment, the antibody comprises an HCVR having the amino acid sequence of SEQ ID NO:18 with 8 or fewer conservative amino acid substitutions. In another embodiment, the antibody comprises an HCVR having the amino acid sequence of SEQ ID NO:18 with 6 or fewer conservative amino acid substitutions. In another embodiment, the antibody comprises an HCVR having the amino acid sequence of SEQ ID NO:18 with 4 or fewer conservative amino acid substitutions. In another embodiment, the antibody comprises an HCVR having the amino acid sequence of SEQ ID NO:18 with 2 or fewer conservative amino acid substitutions. In one embodiment, the antibody comprises an LCVR having the amino acid sequence of SEQ ID NO: 20 with 8 or fewer conservative amino acid substitutions. In another embodiment, the antibody comprises an LCVR having the amino acid sequence of SEQ ID NO: 20 with 6 or fewer conservative amino acid substitutions. In another embodiment, the antibody comprises an LCVR having the amino acid sequence of SEQ ID NO:20 with 4 or fewer conservative amino acid substitutions. In another embodiment, the antibody comprises an LCVR having the amino acid sequence of SEQ ID NO:20 with 2 or fewer conservative amino acid substitutions.

The term “surface plasmon resonance”, as used herein, refers to an optical phenomenon that allows for the analysis of real-time interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore™ system (Biacore Life Sciences division of GE Healthcare, Piscataway, N.J.).

The term “K_(D)”, as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

The term “epitope” refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstance, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.

The term “substantial identity” or “substantially identical,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

As applied to polypeptides, the term “substantial similarity” or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

Preparation of Human Antibodies

Methods for generating monoclonal antibodies, including fully human monoclonal antibodies are known in the art. Any such known methods can be used in the context of the present invention to make human antibodies that specifically bind to human Ang-2 and which possess one or more of the antigen-binding and/or functional characteristics of any of the exemplary anti-Ang-2 antibodies disclosed herein.

Using VELOCIMMUNE™ technology or any other known method for generating monoclonal antibodies, high affinity chimeric antibodies to Ang-2 are initially isolated having a human variable region and a mouse constant region. As in the experimental section below, the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate the fully human antibody of the invention, for example wild-type or modified IgG1 or IgG4. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.

Bioequivalents

The anti-Ang-2 antibodies and antibody fragments of the present invention encompass proteins having amino acid sequences that vary from those of the described antibodies, but that retain the ability to bind human Ang-2. Such variant antibodies and antibody fragments comprise one or more additions, deletions, or substitutions of amino acids when compared to parent sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies. Likewise, the anti-Ang-2 antibody-encoding DNA sequences of the present invention encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequence, but that encode an anti-Ang-2 antibody or antibody fragment that is essentially bioequivalent to an anti-Ang-2 antibody or antibody fragment of the invention. Examples of such variant amino acid and DNA sequences are discussed above.

Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single does or multiple dose. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied.

In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.

Bioequivalence may be demonstrated by in vivo and in vitro methods. Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antibody.

Bioequivalent variants of anti-Ang-2 antibodies of the invention may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation.

Biological and Therapeutic Characteristics of the Antibodies

In general, the antibodies of the instant invention bind to human Ang-2 with a K_(D) of less than 100 pM, typically with a K_(D) of less than 50 pM, and in certain embodiments, with a K_(D) of less than 40 pM, when measured by binding to antigen either immobilized on solid phase or in solution phase.

In addition, certain exemplary anti-Ang-2 antibodies of the invention may exhibit one or more of the following characteristics: (1) ability to bind to human Ang-2 but not to mouse Ang-2; (2) ability to bind to human Ang-2 and to mouse Ang-2; (3) ability to bind to human Ang-2 but not to human Ang-1, -3 or -4; (4) ability to bind to human Ang-2 but not to mouse Ang-1, -3 or -4; (5) ability to bind to human Ang-2 and to human Ang-1, -3 or -4; (6) ability to bind to human Ang-2 and to mouse Ang-1, -3 or -4; (7) ability to block binding of human Ang-2 to human Tie-2; (8) ability to block binding of human Ang-2 to mouse Tie-2; (9) ability to block binding of mouse Ang-2 to human Tie-2; (10) ability to block binding of mouse Ang-2 to mouse Tie-2; (11) ability to block binding of human Ang-1 to human Tie-2; (12) ability to block binding of human Ang-1 to mouse Tie-2; (13) ability to block binding of mouse Ang-1 to human Tie-2; (14) ability to block binding of mouse Ang-1 to mouse Tie-2; (15) ability to inhibit human Ang-2-induced phosphorylation of human Tie-2; (16) ability to inhibit human Ang-2-induced phosphorylation of mouse Tie-2; (17) ability to inhibit mouse Ang-2-induced phosphorylation of human Tie-2; (18) ability to inhibit mouse Ang-2 induced phosphorylation of mouse Tie-2; (19) ability to inhibit human Ang-1-induced phosphorylation of human Tie-2; (20) ability to inhibit human Ang-1-induced phosphorylation of mouse Tie-2; (21) ability to inhibit mouse Ang-1-induced phosphorylation of human Tie-2; (22) ability to inhibit mouse-Ang-1-induced phosphorylation of mouse Tie-2; (23) ability to inhibit in vivo angiogenesis in an experimental model (e.g., angiogenesis induced by a Matrigel plug containing MCF-7 cells implanted subcutaneously into nude mice); and/or (24) ability to inhibit or decrease tumor volume in a mouse xenograft model.

The present invention also includes antibodies that bind with high affinity to a construct comprising the Ang-2 fibronectin-like domain but lacking the Ang-2 N-terminal coiled-coil domain (such constructs are referred to herein as “Ang-2FD”). Exemplary Ang-2FD constructs include human Ang-2FD (SEQ ID NO:519), mouse Ang-2FD (SEQ ID NO:520), and monkey Ang-2FD (SEQ ID NO:521). The human, mouse and monkey Ang-2FD constructs may be monomeric or dimeric. Ang-2FD constructs may also include other non-Ang-2 amino acid sequences such as a human or mouse Fc domain linked to the Ang-2FD molecules. Another exemplary Ang-2FD construct is referred to herein as “hBA2” (or human “bow-Ang2”) which is a tetramer of human Ang-2 fibrinogen-like domains associated with one another via a human or mouse Fc domain to form a bow-tie-like configuration. Typically, hBA2 consists of two Ang-2 dimers, wherein each Ang-2 dimer contains two Ang-2 fibronectin-like domains connected to one another via an Fc domain. Exemplary hBA2 components include the polypeptides designated hBA2-hIgG1 (SEQ ID NO:522) and hBA2-mIgG2a (SEQ ID NO:523). Unexpectedly, certain anti-Ang-2 antibodies of the present invention were found to bind to Ang-2FD constructs with much higher affinities than an known Ang-2 control antibody (see Examples set forth herein).

High affinity binding, in the context of anti-Ang-2 antibody binding to a human or mouse dimeric Ang-2FD construct, means that the anti-Ang-2 antibody binds the human or mouse dimeric Ang-2FD with a K_(D) of less than 300 pM. For example, anti-Ang-2 antibodies that bind with high affinity to human or mouse dimeric Ang-2FD include antibodies that bind to human or mouse dimeric Ang-2-FD with a K_(D) of less than 300 pM, less than 250 pM, less than 200 pM, less than 190 pM, less than 180 pM, less than 170 pM, less than 160 pM, less than 150 pM, less than 140 pM, less than 130 pM, less than 120 pM, less than 110 pM, less than 100 pM, less than 90 pM, less than 80 pM, less than 70 pM, less than 60 pM or less than 50 pM, as measured at 25° C. in a surface Plasmon resonance assay.

High affinity binding, in the context of anti-Ang-2 antibody binding to a monkey dimeric Ang-2FD construct, means that the anti-Ang-2 antibody binds the monkey dimeric Ang-2FD with a K_(D) of less than 500 pM. For example, anti-Ang-2 antibodies that bind with high affinity to monkey dimeric Ang-2FD include antibodies that bind to monkey Ang-2-FD with a K_(D) of less than 500 pM, less than 450 pM, less than 400 pM, less than 350 pM, less than 300 pM, less than 250 pM, less than 200 pM, less than 190 pM, less than 180 pM, less than 170 pM, less than 160 pM, less than 150 pM, less than 140 pM, less than 130 pM, less than 120 pM, less than 110 pM, less than 100 pM, less than 90 pM, or less than 80 pM, as measured at 25° C. in a surface Plasmon resonance assay.

High affinity binding, in the context of anti-Ang-2 antibody binding to a human monomeric Ang-2FD construct, means that the anti-Ang-2 antibody binds the human monomeric Ang-2FD with a K_(D) of less than 40 nM. For example, anti-Ang-2 antibodies that bind with high affinity to human monomeric Ang-2FD include antibodies that bind to human monomeric Ang-2-FD with a K_(D) of less than 40 nM, less than 30 nM, less than 25 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.9 nM, less than 0.8 nM, less than 0.7 nM, or less than 0.6 nM as measured at 25° C. in a surface Plasmon resonance assay.

High affinity binding, in the context of anti-Ang-2 antibody binding to a hBA2 construct, means that the anti-Ang-2 antibody binds the hBA2 with a K_(D) of less than 80 pM. For example, anti-Ang-2 antibodies that bind with high affinity to hBA2 include antibodies that bind to hBA2 with a K_(D) of less than 80 pM, less than 75 pM, less than 70 pM, less than 65 pM, less than 60 pM, less than 55 pM, less than 50 pM, less than 45 pM, less than 40 pM, less than 35 pM, less than 30 pM, less than 25 pM, less than 20 pM, less than 18 pM, less than 16 pM, less than 14 pM, or less than 12 pM, as measured at 25° C. in a surface Plasmon resonance assay.

The present invention includes antibodies that bind Ang-2 but do not substantially bind Ang-1. As used herein, an antibody “does not substantially bind Ang-1” if the antibody, when tested for binding to Ang-1 in a surface plasmon resonance assay in which the antibody is captured on a surface and full-length wild-type human Ang-1 at a concentration of about 25 nM is injected over the captured antibody surface at a flowrate of about 60 μl/min for about 3 minutes at 25° C., exhibits a K_(D) of greater than about 1 nM, e.g., a K_(D) of greater than about 5 nM, greater than about 10 nM, greater than about 50 nM, greater than about 100 nM, greater than about 150 nM, greater than about 200 nM, greater than about 250 nM, greater than about 300 nM, greater than about 350 nM, greater than about 400 nM, greater than about 450 nM, greater than about 500 nM, or more. (See, e.g., Example 4). In addition, an antibody “does not substantially bind Ang-1” if the antibody fails to exhibit any binding to Ang-1 when tested in such an assay or equivalent thereof.

The present invention also includes antibodies that block the binding of Ang-2 to Tie-2 but do not substantially block the binding of Ang-1 to Tie-2. As used herein, an antibody “does not substantially block the binding of Ang-1 to Tie-2” if, when the antibody is premixed with Ang-1 antigen at a ratio of about 100:1 (antibody:antigen) and allowed to incubate at 25° C. for about 60 minutes and then the equilibrated mixture is tested for binding to Tie-2 by surface plasmon resonance over a Tie-2-coated surface (5 μl/min for 5 min. at 25° C.), the amount of Ang-1 bound to Tie-2 is at least 50% the amount of Ang-1 bound to Tie-2 in the presence of an irrelevant control molecule. (See, e.g., Example 6). For example, if the amount of Ang-1 bound to Tie-2 following preincubation with an antibody is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% the amount of Ang-1 that binds to Tie-2 following preincubation with an irrelevant control molecule under the above noted experimental conditions, then the antibody is deemed to “not substantially block the binding of Ang-1 to Tie-2.”

Moreover, the present invention includes antibodies that block or substantially attenuate a biological activity of Ang-2 (e.g., Ang-2-mediated phosphorylation of Tie-2; Ang-2-induced angiogenesis; etc.) but do not block or substantially attenuate the corresponding biological activity of Ang-1 (e.g., Ang-1-mediated phosphorylation of Tie-2; Ang-1-induced angiogenesis; etc). Assays and tests useful for determining whether an antibody satisfies one or more of the characteristics listed above will be readily known and easily practiced by persons of ordinary skill in the art and/or can be fully ascertained from the present disclosure. For example, the experimental procedures detailed below can be used to determine whether a given antibody binds or does not bind to Ang-2 and/or Ang-1; blocks or does not block binding of Ang-2 and/or Ang-1 to Tie-2; inhibits or does not inhibit Ang-2- and/or Ang-1-mediated phosphorylation of Tie-2; etc.

Epitope Mapping and Related Technologies

To screen for antibodies that bind to a particular epitope (e.g., those which block binding of IgE to its high affinity receptor), a routine cross-blocking assay such as that described “Antibodies,” Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., N.Y.) can be performed. Other methods include alanine scanning mutants, peptide blots (Reineke (2004) Methods Mol Biol 248:443-63), or peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer (2000) Protein Science 9: 487-496).

The term “epitope” refers to a site on an antigen to which B and/or T cells respond. B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.

Modification-Assisted Profiling (MAP), also known as Antigen Structure-based Antibody Profiling (ASAP) is a method that categorizes large numbers of monoclonal antibodies (mAbs) directed against the same antigen according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (US 2004/0101920). Each category may reflect a unique epitope either distinctly different from or partially overlapping with epitope represented by another category. This technology allows rapid filtering of genetically identical antibodies, such that characterization can be focused on genetically distinct antibodies. When applied to hybridoma screening, MAP may facilitate identification of rare hybridoma clones that produce mAbs having the desired characteristics. MAP may be used to sort the anti-Ang-2 antibodies of the invention into groups of antibodies binding different epitopes.

Anti-Ang-2 antibodies can bind to an epitope within the amino-terminal coiled-coil domain or within the carboxy-terminal fibrinogen-like domain (“FD”). In preferred embodiments of the present invention, the anti-Ang-2 antibodies and antigen binding fragments thereof bind to an epitope within the FD.

The amino acids within the FD of Ang-2 that interact with Tie-2 have been ascertained from crystal structure analysis. See Barton et al., Nat. Struct. Mol. Biol. 13:524-532 (May 2006). With regard to antibodies that block the binding of Ang-2 to Tie-2 but do not substantially block binding of Ang-1 to Tie-2 (e.g., H1H685P, see Examples 5 and 6 below), the epitope to which such antibodies bind may include one or more amino acids of Ang-2 that (a) interact with Tie-2 and (b) are non-identical to the corresponding amino acid in Ang-1. (See FIG. 1). Thus, the epitope to which such Ang-2 preferential antibodies bind may include one or more of the following amino acids of hAng-2 (SEQ ID NO:518): S-417; K-432; I-434; N-467; F-469; Y-475; or S-480. For example, the present inventors have discovered that antibodies which interact with amino acids F-469, Y-475, and S-480 of Ang-2 (SEQ ID NO:518) preferentially interact with Ang-2 over Ang-1, and this preferential binding may have therapeutic benefits. Thus, the present invention includes anti-Ang-2 antibodies which specifically bind human angiopoietin-2 (hAng-2) but do not substantially bind hAng-1, wherein the antibodies bind an epitope on hAng-2 (SEQ ID NO:518) comprising amino acids F-469, Y-475, and S-480. Similarly, the present invention includes anti-Ang-2 antibodies which block the binding of hAng-2 to hTie-2 but do not substantially block the binding of hAng-1 to hTie-2, wherein the antibodies bind an epitope on hAng-2 (SEQ ID NO:518) comprising amino acids F-469, Y-475, and S-480.

The present invention includes anti-Ang-2 antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein (e.g., H1H685, H1H690, H1H691, H1H696, H1H706, H1M724 and/or H2M744). Likewise, the present invention also includes anti-Ang-2 antibodies that compete for binding to Ang-2 with any of the specific exemplary antibodies described herein (e.g., H1H685, H1H690, H1H691, H1H696, H1H706, H1M724 and/or H2M744).

One can easily determine whether an antibody binds to the same epitope as, or competes for binding with, a reference anti-Ang-2 antibody by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope as a reference anti-Ang-2 antibody of the invention, the reference antibody is allowed to bind to an Ang-2 protein or peptide under saturating conditions. Next, the ability of a test antibody to bind to the Ang-2 molecule is assessed. If the test antibody is able to bind to Ang-2 following saturation binding with the reference anti-Ang-2 antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-Ang-2 antibody. On the other hand, if the test antibody is not able to bind to the Ang-2 molecule following saturation binding with the reference anti-Ang-2 antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference anti-Ang-2 antibody of the invention. Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, Biacore, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art. In accordance with certain embodiments of the present invention, two antibodies bind to the same (or overlapping) epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antibodies are deemed to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies are deemed to have “overlapping epitopes” if only a subset of the amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

To determine if an antibody competes for binding with a reference anti-Ang-2 antibody, the above-described binding methodology is performed in two orientations: In a first orientation, the reference antibody is allowed to bind to an Ang-2 molecule under saturating conditions followed by assessment of binding of the test antibody to the Ang-2 molecule. In a second orientation, the test antibody is allowed to bind to an Ang-2 molecule under saturating conditions followed by assessment of binding of the reference antibody to the Ang-2 molecule. If, in both orientations, only the first (saturating) antibody is capable of binding to the Ang-2 molecule, then it is concluded that the test antibody and the reference antibody compete for binding to Ang-2. As will be appreciated by a person of ordinary skill in the art, an antibody that competes for binding with a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.

Species Selectivity and Species Cross-Reactivity

According to certain embodiments of the invention, the anti-Ang-2 antibodies bind to human Ang-2 but not to Ang-2 from other species. Alternatively, the anti-Ang-2 antibodies of the invention, in certain embodiments, bind to human Ang-2 and to Ang-2 from one or more non-human species. For example, the Ang-2 antibodies of the invention may bind to human Ang-2 and may bind or not bind, as the case may be, to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomologous, marmoset, rhesus or chimpanzee Ang-2.

Immunoconjugates

The invention encompasses anti-Ang-2 monoclonal antibodies conjugated to a therapeutic moiety (“immunoconjugate”), such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioisotope. Cytotoxic agents include any agent that is detrimental to cells. Examples of suitable cytotoxic agents and chemotherapeutic agents for forming immunoconjugates are known in the art, see for example, WO 05/103081).

Multispecific Antibodies

The antibodies of the present invention may be monospecific, bispecific, or multispecific. Multispecific mAbs may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al. (1991) J. Immunol. 147:60-69. The anti-Ang-2 antibodies of the present invention, or portions thereof, can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein, to form a multispecific molecule. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or a multispecific antibody with a second binding specificity.

An exemplary bi-specific antibody format that can be used in the context of the present invention involves the use of a first immunoglobulin (Ig) C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first and second Ig C_(H)3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference. In one embodiment, the first Ig C_(H)3 domain binds Protein A and the second Ig C_(H)3 domain contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second C_(H)3 may further comprise a Y96F modification (by IMGT; Y436F by EU). Further modifications that may be found within the second C_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1 antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the case of IgG4 antibodies. Variations on the bi-specific antibody format described above are contemplated within the scope of the present invention.

Therapeutic Uses of the Antibodies

The antibodies of the invention are useful, inter alia, for the treatment, prevention and/or amelioration of any disease or disorder associated with Ang-2 activity, including diseases or disorders associated with angiogenesis. The antibodies and antigen-binding fragments of the present invention may be used to treat, e.g., primary and/or metastatic tumors arising in the brain and meninges, oropharynx, lung and bronchial tree, gastrointestinal tract, male and female reproductive tract, muscle, bone, skin and appendages, connective tissue, spleen, immune system, blood forming cells and bone marrow, liver and urinary tract, and special sensory organs such as the eye. In certain embodiments, the antibodies and antigen-binding fragments of the invention are used to treat one or more of the following cancers: renal cell carcinoma, pancreatic carcinoma, breast cancer, prostate cancer, malignant gliomas, osteosarcoma, colorectal cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, or melanoma.

For example, the present invention includes methods of treating advanced cancer, the methods comprising administering to a subject in need thereof an isolated antibody or antigen binding fragment thereof that binds specifically to Ang-2, but does not bind specifically to Ang-1, as described herein. Embodiments of the invention pertain to methods of treating advanced solid tumors such as advanced ovarian cancer, colorectal cancer or hepatocellular carcinoma. In some embodiments, the methods comprise administering a second therapeutic agent in combination with an anti-Ang-2 antibody. In one embodiment, the second therapeutic agent may be a VEGF antagonist (such as aflibercept, a recombinant fusion protein consisting of VEGF receptor extracellular domains fused to the Fc portion of human IgG1).

In certain embodiments, the present invention includes methods to inhibit angiogenesis, the methods comprising administering to a subject in need thereof an isolated antibody or antigen binding fragment thereof that binds specifically to Ang-2, but does not bind specifically to Ang-1. In some embodiments, the methods include administering a second angiogenesis inhibitor. The second angiogenesis inhibitor may be another antibody or a recombinant protein such as a VEGF antagonist (e.g., aflibercept).

The present invention includes methods to treat advanced cancer in patients who are refractory to standard or conventional anti-tumor therapy. For example, the present methods may be used in patients suffering from platinum-resistant ovarian cancer.

The antibodies and antigen-binding fragments of the present invention may also be useful for the treatment of one or more eye disorders. Exemplary eye disorders that can be treated with the antibodies and antigen-binding fragments of the invention include, e.g., age-related macular degeneration, diabetic retinopathy, and other eye disorders associated with choroidal neovascularization, vascular leak, retinal edema and inflammation. Additionally, the anti-Ang-2 antibodies of the invention may be administered as an adjuvant to glaucoma surgery to prevent early hem- and lymphangiogenesis and macrophage recruitment to the filtering bleb after glaucoma surgery, and improve clinical outcome.

In other embodiments of the present invention, the antibodies or antigen-binding fragments are used to treat hypertension, diabetes (including non-insulin dependent diabetes mellitus), psoriasis, arthritis (including rheumatoid arthritis), asthma, sepsis, kidney disease and edema associated with injury, stroke or tumor.

Ang-2 expression has been shown to correlate with the severity of various inflammatory and/or infectious diseases (see, e.g., Siner et al., 2009, Shock 31:348-353; Yeo et al., 2008, Proc. Natl. Acad. Sci. (USA):105:17097-17102). Accordingly, the anti-Ang-2 antibodies of the present invention can be used to treat, prevent or ameliorate one or more inflammatory or infectious diseases. Exemplary infectious diseases that can be treated, prevented or ameliorated by administration of the anti-Ang-2 antibodies of the invention include, but are not limited to: malaria (Plasmodium falciparum infection), viral hemorrhagic fevers (e.g., dengue fever), rickettsial infection, toxic shock syndrome, sepsis, hepatitis C, Bartonella bacilliformis infection, leishmaniasis, mycobacterial infection, and Epstein-Barr virus infection

Therapeutic Formulation and Administration

The invention provides therapeutic compositions comprising the anti-Ang-2 antibodies or antigen-binding fragments thereof of the present invention. In certain embodiments, the invention provides for methods of treatment which comprise administering an anti-Ang-2 antibody to a patient, wherein the anti-Ang-2 antibody is contained within a pharmaceutical composition. The therapeutic compositions in of the present invention may further comprise one or more pharmaceutically acceptable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like (herein collectively referred to as “pharmaceutically acceptable carriers or diluents”). A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA, 1998, J Pharm Sci Technol 52:238-311.

The dose of antibody according to methods of the present invention may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like. The preferred dose is typically calculated according to body weight or body surface area. When an antibody of the present invention is used for treating a condition or disease associated with Ang-2 activity in an adult patient, it may be advantageous to intravenously administer the antibody of the present invention normally at a single dose of about 0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. Effective dosages and schedules for administering Ang-2 antibodies may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly. Moreover, interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al. 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.

A pharmaceutical composition of the present invention can be delivered, e.g., subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but certainly are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but certainly are not limited to the SOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly).

For the treatment of eye disorders, the antibodies and antigen-binding fragments of the invention may be administered, e.g., by eye drops, subconjunctival injection, subconjunctival implant, intravitreal injection, intravitreal implant, sub-Tenon's injection or sub-Tenon's implant.

The pharmaceutical composition can be also delivered in a vesicle, in particular a liposome (see Langer 1990 Science 249:1527-1533; Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton 1987 CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138).

The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforesaid antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the aforesaid antibody is contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms.

Dosage

The amount of anti-Ang-2 antibody administered to a subject according to the methods of the present invention is, generally, a therapeutically effective amount. As used herein, the phrase “therapeutically effective amount” means an amount of anti-Ang-2 antibody that results in one or more of: (a) regression of a tumor or inhibition of angiogenesis in a tumor; and/or (b) a detectable improvement in one or more symptoms or indicia of hepatic function or renal function or bone marrow function (as defined elsewhere herein). A “therapeutically effective amount” also includes an amount of anti-Ang-2 antibody that inhibits, prevents, lessens, or delays the progression of a tumor in a subject.

In the case of an anti-Ang-2 antibody, a therapeutically effective amount can be from about 0.1 mg to about 1500 mg, e.g., about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, or about 1500 mg, of the anti-Ang-2 antibody.

The amount of anti-Ang-2 antibody contained within the individual doses may be expressed in terms of milligrams of antibody per kilogram of patient body weight (i.e., mg/kg). For example, the anti-Ang-2 antibody may be administered to a patient at a dose of about 0.0001 to about 20 mg/kg of patient body weight. In certain embodiments, the anti-Ang-2 antibody may be administered at a dose of about 1, 3, 6, 12 or 20 mg/kg of patient body weight.

Combination Therapies

The methods of the present invention, according to certain embodiments, comprise administering to the subject one or more additional therapeutic agents in combination with an anti-Ang-2 antibody. As used herein, the expression “in combination with” means that the additional therapeutic agents are administered before, after, or concurrent with the pharmaceutical composition comprising the anti-Ang-2 antibody. The term “in combination with” also includes sequential or concomitant administration of anti-Ang-2 antibody and a second therapeutic agent.

For example, when administered “before” the pharmaceutical composition comprising the anti-Ang-2 antibody, the additional therapeutic agent may be administered about 72 hours, about 60 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 30 minutes, about 15 minutes or about 10 minutes prior to the administration of the pharmaceutical composition comprising the anti-Ang-2 antibody. When administered “after” the pharmaceutical composition comprising the anti-Ang-2 antibody, the additional therapeutic agent may be administered about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours or about 72 hours after the administration of the pharmaceutical composition comprising the anti-Ang-2 antibody. Administration “concurrent” or with the pharmaceutical composition comprising the anti-Ang-2 antibody means that the additional therapeutic agent is administered to the subject in a separate dosage form within less than 5 minutes (before, after, or at the same time) of administration of the pharmaceutical composition comprising the anti-Ang-2 antibody, or administered to the subject as a single combined dosage formulation comprising both the additional therapeutic agent and the anti-Ang-2 antibody.

Combination therapies may include an anti-Ang-2 antibody of the invention and, for example, another Ang-2 antagonist (e.g., an anti-Ang-2 antibody, peptibody, or CovX-body such as CVX-060 (see U.S. Pat. No. 7,521,425)). Other exemplary additional therapeutic agents that may be combined with or administered in combination with an anti-Ang-2 antibody of the present invention include, e.g., an EGFR antagonist (e.g., an anti-EGFR antibody [e.g., cetuximab or panitumumab] or small molecule inhibitor of EGFR [e.g., gefitinib or erlotinib]), an antagonist of another EGFR family member such as Her2/ErbB2, ErbB3 or ErbB4 (e.g., anti-ErbB2, anti-ErbB3 or anti-ErbB4 antibody or small molecule inhibitor of ErbB2, ErbB3 or ErbB4 activity), an antagonist of EGFRvIII (e.g., an antibody that specifically binds EGFRvIII), a cMET anagonist (e.g., an anti-cMET antibody), an IGF1R antagonist (e.g., an anti-IGF1R antibody), a B-raf inhibitor (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720), a PDGFR-α inhibitor (e.g., an anti-PDGFR-α antibody), a PDGFR-β inhibitor (e.g., an anti-PDGFR-β antibody), a VEGF antagonist (e.g., aflibercept, a VEGF-Trap, see, e.g., U.S. Pat. No. 7,087,411 (also referred to herein as a “VEGF-inhibiting fusion protein”), anti-VEGF antibody (e.g., bevacizumab), a small molecule kinase inhibitor of VEGF receptor (e.g., sunitinib, sorafenib or pazopanib)), a DLL4 antagonist (e.g., an anti-DLL4 antibody disclosed in US 2009/0142354 such as REGN421), a FOLH1 antagonist (e.g., an anti-FOLH1 antibody), a PRLR antagonist (e.g., an anti-PRLR antibody), a STEAP1 or STEAP2 antagonist (e.g., an anti-STEAP1 antibody or an anti-STEAP2 antibody), a TMPRSS2 antagonist (e.g., an anti-TMPRSS2 antibody), a MSLN antagonist (e.g., an anti-MSLN antibody), a CA9 antagonist (e.g., an anti-CA9 antibody), a uroplakin antagonist (e.g., an anti-uroplakin antibody), a monovalent CD20 antagonist (e.g., a monovalent anti-CD20 antibody such as rituximab), etc. Other agents that may be beneficially administered in combination with the anti-Ang-2 antibodies of the invention include cytokine inhibitors, including small-molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or to their respective receptors.

The present invention also includes therapeutic combinations comprising any of the anti-Ang-2 antibodies mentioned herein and an inhibitor of one or more of VEGF, DLL4, EGFR, or any of the aforementioned cytokines, wherein the inhibitor is an aptamer, an antisense molecule, a ribozyme, an siRNA, a peptibody, a nanobody or an antibody fragment (e.g., Fab fragment; F(ab′)₂ fragment; Fd fragment; Fv fragment; scFv; dAb fragment; or other engineered molecules, such as diabodies, triabodies, tetrabodies, minibodies and minimal recognition units). The anti-Ang-2 antibodies of the invention may also be administered in combination with antivirals, antibiotics, analgesics, corticosteroids and/or NSAIDs. The anti-Ang-2 antibodies of the invention may also be administered as part of a treatment regimen that also includes radiation treatment and/or conventional chemotherapy. When combined with one or more additional agents, the anti-Ang-2 antibodies of the invention may be administered prior to, simultaneous with (e.g., in the same formulation or in separate formulations), or subsequent to the administration of the other agent(s).

The methods of the invention comprise administering an anti-Ang-2 antibody in combination with a second therapeutic agent for additive or synergistic activity to treat advanced solid tumor malignancy.

Administration Regimens

The present invention includes methods comprising administering to a subject a pharmaceutical composition comprising an anti-Ang-2 antibody at a dosing frequency of about four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or less frequently so long as a therapeutic response is achieved. In certain embodiments involving the administration of a pharmaceutical composition comprising an anti-Ang-2 antibody, once in 2 weeks dosing at an amount of about 1, 3, 6 or 12 mg/kg of patient body weight, can be employed.

According to certain embodiments of the present invention, multiple doses of an anti-Ang-2 antibody may be administered to a subject over a defined time course. The methods according to this aspect of the invention comprise sequentially administering to a subject multiple doses of an anti-Ang-2 antibody. As used herein, “sequentially administering” means that each dose of anti-Ang-2 antibody is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). The present invention includes methods which comprise sequentially administering to the patient a single initial dose of an anti-Ang-2 antibody, followed by one or more secondary doses of the anti-Ang-2 antibody, and optionally followed by one or more tertiary doses of the anti-Ang-2 antibody.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” refer to the temporal sequence of administration of the anti-Ang-2 antibody. Thus, the “initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”); the “secondary doses” are the doses which are administered after the initial dose; and the “tertiary doses” are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of anti-Ang-2 antibody, but generally may differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of anti-Ang-2 antibody contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, one or more (e.g., 1, 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as “loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g., “maintenance doses”). For example, an anti-Ang-2 antibody may be administered to a patient with advanced malignancy at a loading dose of about 600 mg or about 1200 mg followed by one or more maintenance doses of about 60 mg to about 360 mg.

In one exemplary embodiment of the present invention, each secondary and/or tertiary dose is administered 1 to 14 (e.g., 1, 1½, 2, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13, 13½, 14, 14½, or more) weeks after the immediately preceding dose. The phrase “the immediately preceding dose,” as used herein, means, in a sequence of multiple administrations, the dose of anti-Ang-2 antibody which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.

The methods according to this aspect of the invention may comprise administering to a patient any number of secondary and/or tertiary doses of an anti-Ang-2 antibody. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.

The present invention includes methods comprising sequential administration of an anti-Ang-2 antibody and a second therapeutic agent, to a patient to treat advanced cancer. In some embodiments, the present methods comprise administering one or more doses of an anti-Ang-2 antibody followed by one or more doses of a second therapeutic agent. For example, one or more doses of about 1 mg/kg to about 12 mg/kg of patient body weight of anti-Ang-2 antibody may be administered after which one or more doses of a second therapeutic agent (e.g., aflibercept or any other therapeutic agent, as described elsewhere herein) may be administered to treat, alleviate, reduce or ameliorate one or more conditions associated with advanced cancer (e.g., angiogenesis inhibition). In some embodiments, the anti-Ang-2 antibody is administered at one or more doses resulting in an improvement in one or more bone marrow function- or hepatic function-associated parameters followed by the administration of a second therapeutic agent to prevent recurrence or have additive activity. Alternative embodiments of the invention pertain to concomitant administration of anti-Ang-2 antibody are administered and a second therapeutic agent is administered at a separate dosage at a similar or different frequency relative to the anti-Ang-2 antibody. In some embodiments, the second therapeutic agent is administered before, after or concurrently with the anti-Ang-2 antibody.

Treatment Populations

The present invention includes methods which comprise administering to a subject in need thereof a therapeutic composition comprising an isolated antibody or antigen-binding fragment thereof that binds specifically to Ang-2 but not specifically to Ang-1, as described herein. As used herein, the expression “a subject in need thereof” means a human or non-human animal that exhibits one or more symptoms or indicia of advanced solid tumor malignancy, and/or who has been diagnosed with advanced cancer. In certain embodiments, the methods of the present invention may be used to treat patients that show altered levels of one or more cancer-associated parameters or a diagnosis of advanced malignancy by histology, cytology or radiologic confirmation. For example, the methods of the present invention comprise administering an anti-Ang-2 antibody to patients with elevated levels of calculated creatinine clearance.

In the context of the present invention, “a subject in need thereof” may include, e.g., subjects who, prior to treatment, exhibit (or have exhibited) one or more biochemical parameters of bone marrow function (e.g, a high hemoglobin, absolute neutrophil count, platelet count), or hepatic function (e.g., total bilirubin, alkaline phosphatase) or renal function (e.g., serum creatinine, calculated creatinine clearance). In some embodiments, “a subject in need thereof” may include patients with confirmed diagnosis of adenocarcinoma by histology or cytology or by radiologic confirmation. In some embodiments, the methods may be used to treat a subject whose performance status as a cancer patient is assessed by one of the performance scores known in the art, e.g., the Eastern Cooperative Oncology Group (ECOG) performance score of 0 or 1. The ECOG performance status scores are given in the following table:

Grade Description 0 Fully active, able to carry on all pre-disease performance without restriction 1 Restricted in physically strenuous activity but ambulatory and able to carry out work of a light or sedentary nature, e.g., light house work, office work 2 Ambulatory and capable of all self-care but unable to carry out any work activities. Up and about more than 50% of waking hours 3 Capable of limited self-care, confined to bed or chair more than 50% of waking hours 4 Completely disabled. Cannot carry on any self-care. Totally confined to bed or chair 5 Dead

The present invention includes methods to treat adult patients with advanced solid tumor malignancies. In some embodiments, the methods are used to treat patients with advanced ovarian cancer, advanced hepatocellular carcinoma or colorectal cancer. In some embodiments, the present methods are used for patients with confirmed diagnosis of adenocarcinoma of the ovary, fallopian tubes or primary peritoneal cancer, metastatic colorectal adenocarcinoma or advanced hepatocellular cancer.

The present invention includes methods to treat advanced cancer in patients resistant, non-responsive or inadequately responsive to standard or conventional anti-cancer therapy. The term “resistant, non-responsive or inadequately responsive to standard or conventional anti-cancer therapy”, as used herein, refers to subjects or patients with cancer who have been treated with an anti-cancer agent known in the art and wherein said agent does not have a therapeutic effect. For example, the present methods may be used to treat patients with advanced platinum-resistant ovarian cancer. In some embodiments, the term refers to reduced patient compliance and/or toxicity and side effects and/or ineffectiveness of the administered anti-cancer agent to reduce or inhibit or delay tumor progression or kill tumor cells. In some embodiments, the term refers to patients suffering from advanced cancer who are refractory to treatment by an anti-cancer agent known in the art. In some embodiments, the term refers to patients suffering from cancer wherein there is tumor growth or disease progression while being treated with a conventional agent. In one embodiment, the methods of the present invention are used to treat platinum-resistant or refractory cancer disease wherein there is disease recurrence within 6 months of last dose of platinum- or disease progression while being treated with platinum-based regimen. In some embodiments, the patients who are “resistant, non-responsive or inadequately responsive to standard or conventional anti-cancer therapy” may show no improvement in one or more parameters of bone marrow function or hepatic function or renal function. Examples of such parameters are described elsewhere herein. For example, treatment with a conventional anti-cancer agent may result in no improvement in hepatic function as characterized by total bilirubin.

Diagnostic Uses of the Antibodies

The anti-Ang-2 antibodies of the present invention may also be used to detect and/or measure Ang-2 in a sample, e.g., for diagnostic purposes. For example, an anti-Ang-2 antibody, or fragment thereof, may be used to diagnose a condition or disease characterized by aberrant expression (e.g., over-expression, under-expression, lack of expression, etc.) of Ang-2. Exemplary diagnostic assays for Ang-2 may comprise, e.g., contacting a sample, obtained from a patient, with an anti-Ang-2 antibody of the invention, wherein the anti-Ang-2 antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-Ang-2 antibody can be used in diagnostic applications in combination with a secondary antibody which is itself detectably labeled. The detectable label or reporter molecule can be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure Ang-2 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1. Generation of Human Antibodies to Human Ang-2

Human Ang-2 antigen was administered directly, with an adjuvant to stimulate the immune response, to a VELOCIMMUNE® mouse comprising DNA encoding human Immunoglobulin heavy and kappa light chain variable regions. The antibody immune response was monitored by an Ang-2-specific immunoassay. When a desired immune response was achieved splenocytes were harvested and fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines. The hybridoma cell lines were screened and selected to identify cell lines that produce Ang-2-specific antibodies. Using this technique several anti-Ang-2 chimeric antibodies (i.e., antibodies possessing human variable domains and mouse constant domains) were obtained; exemplary antibodies generated in this manner were designated as follows: H1M724, H1M727, H1M728, H2M730, H1M732, H1M737, H2M742, H2M743, H2M744, H1M749, H2M750 and H1M810.

Anti-Ang-2 antibodies were also isolated directly from antigen-positive B cells without fusion to myeloma cells, as described in U.S. 2007/0280945A1. Using this method, several fully human anti-Ang-2 antibodies (i.e., antibodies possessing human variable domains and human constant domains) were obtained; exemplary antibodies generated in this manner were designated as follows: H1H685, H1H690, H1H691, H1H693, H1H694, H1H695, H1H696, H1H704, H1H706 and H1H707.

The biological properties of the exemplary anti-Ang-2 antibodies generated in accordance with the methods of this Example are described in detail in the Examples set forth below. The exemplary anti-Ang-2 antibody used in Examples 14 and 15 below is the human anti-Ang-2 antibody designated in Table 2 as H1H685P (also referred to herein as “mAb1”).

Example 2. Variable Gene Utilization Analysis

To analyze the structure of antibodies produced, the nucleic acids encoding antibody variable regions were cloned and sequenced. From the nucleic acid sequence and predicted amino acid sequence of the antibodies, gene usage was identified for each heavy chain variable region (HCVR) and light chain variable region (LCVR) (Table 1).

TABLE 1 HCVR LCVR Antibody Identifier Antibody V_(H) D_(H) J_(H) V_(K) J_(K) HCVR/LCVR SEQ ID NOs H1H685 3-13  3-16 4 3-20 1  2/10 H1H690 3-23 4-4 3 3-11 4 26/34 H1H691 3-9   4-17 6 3-20 4 50/58 H1H693 3-23 4-4 3 1-12 1 74/82 H1H694 3-15 6-6 4 1-5  1  98/106 H1H695 3-33  5-12 6 3-15 5 122/130 H1H696 3-11  4-17 4 1-16 4 146/154 H1H704 3-33 6-6 4 1-16 5 170/178 H1H706 3-33 3-3 3 1-16 1 194/202 H1H707 3-33 3-3 3 3-20 4 218/226 H1M724 3-33 3-3 5 1-17 4 266/274 H1M727 1-18 3-3 6 2-28 2 338/346 H1M728 3-7   6-19 4 1-5  1 290/298 H2M730 3-7   6-13 4 1-5  1 362/370 H1M732 3-15 1-7 4 1-17 3 242/250 H2M742 3-23 5-5 5 2-28 4 386/394 H2M743 3-23 2-8 4 1-12 4 410/418 H2M744 1-18 4-4 5 1-12 4 434/442 H1M749 3-33 5-5 4 3-15 1 314/322 H2M750 3-33 6-6 4 1-16 4 458/466 H1M810 3-23 3-3 3 1-12 1 482/490

Table 2 sets forth the heavy and light chain variable region amino acid sequence pairs of selected anti-Ang-2 antibodies and their corresponding antibody identifiers. The N, P and G designations refer to antibodies having heavy and light chains with identical CDR sequences but with sequence variations in regions that fall outside of the CDR sequences (i.e., in the framework regions). Thus, N, P and G variants of a particular antibody have identical CDR sequences within their heavy and light chain variable regions but contain modifications within the framework regions.

TABLE 2 HCVR/ LCVR HCVR/ HCVR/ SEQ LCVR LCVR Name ID NOs Name SEQ ID NOs Name SEQ ID NOs H1H685N  2/10 H1H685P 18/20 H1H685G 22/24 H1H690N 26/34 H1H690P 42/44 H1H690G 46/48 H1H691N 50/58 H1H691P 66/68 H1H691G 70/72 H1H693N 74/82 H1H693P 90/92 H1H693G 94/96 H1H694N  98/106 H1H694P 114/116 H1H694G 118/120 H1H695N 122/130 H1H695P 138/140 H1H695G 142/144 H1H696N 146/154 H1H696P 162/164 H1H696G 166/168 H1H704N 170/178 H1H704P 186/188 H1H704G 190/192 H1H706N 194/202 H1H706P 210/212 H1H706G 214/216 H1H707N 218/226 H1H707P 234/236 H1H707G 238/240 H1M724N 266/274 H1M724P 282/284 H1M724G 286/288 H1M727N 338/346 H1M727P 354/356 H1M727G 358/360 H1M728N 290/298 H1M728P 306/308 H1M728G 310/312 H2M730N 362/370 H2M730P 378/380 H2M730G 382/384 H1M732N 242/250 H1M732P 258/260 H1M732G 262/264 H1M737N 506/X*  H1M737P 514/X*  H1M737G 516/X*  H2M742N 386/394 H2M742P 402/404 H2M742G 406/408 H2M743N 410/418 H2M743P 426/428 H2M743G 430/432 H2M744N 434/442 H2M744P 450/452 H2M744G 454/456 H1M749N 314/322 H1M749P 330/332 H1M749G 334/336 H2M750N 458/466 H2M750P 474/476 H2M750G 478/480 H1M810N 482/490 H1M810P 498/500 H1M810G 502/504 *The amino acid sequence of the LCVR of H1M737 is not shown. Control Constructs Used in the Following Examples

Various control constructs (anti-Ang-2 antibodies and anti-Ang-2 peptibodies) were included in the following experiments for comparative purposes. The control constructs are designated as follows: Control I: a human anti-Ang-2 antibody with heavy and light chain variable domains having the amino acid sequences of the corresponding domains of “Ab536(THW),” as set forth in US 2006/0018909 (see also Oliner et al., 2004, Cancer Cell 6:507-516); Control II: a peptibody that binds human Ang-2 having the amino acid sequence of “2XCon4(C),” as set forth in U.S. Pat. No. 7,205,275, (see also Oliner et al., 2004, Cancer Cell 6:507-516); Control III: a peptibody that binds human Ang-2 having the amino acid sequence of “L1-7,” as set forth in U.S. Pat. No. 7,138,370; Control IV: a human anti-Ang-2 antibody with heavy and light chain variable regions having the amino acid sequences of the corresponding domains of “3.19.3” as set forth in US 2006/0246071; and Control V: a human anti-Ang-2 antibody with heavy and light chain variable regions having the amino acid sequences of the corresponding domains of “MEDI1/5” as set forth in WO 2009/097325. (Not all control constructs were used in every Example). In the tables that follow, the notations “Ab” and “Pb” are included to identify antibody and peptibody controls, respectively (i.e., Control 1=Ab; Control II=Pb; Control III=Pb; Control IV=Ab; and Control V=Ab).

Example 3. Antigen Binding Affinity Determination

Equilibrium dissociation constants (K_(D) values) for the binding of selected purified Ang-2 antibodies to dimeric fibrinogen-like domain of human (SEQ ID NO: 519), mouse (Mus musculus; SEQ ID NO: 520) and monkey (Macca fascicularis; SEQ ID NO: 521) Ang-2 (Ang-2FD) conjugated to human IgG1 (SEQ ID NO:528) were determined by surface kinetics using a real-time biosensor surface plasmon resonance assay. Antibody was captured on a goat anti-mouse IgG polyclonal antibody surface, a goat anti-human κ polyclonal antibody (Southern Biotech, Birmingham, Ala.) surface or a goat anti-human IgG polyclonal antibody (Jackson Immuno Research Lab, West Grove, Pa.) surface created through direct amine coupling to a BIACORE™ CM5 sensor chip to form a captured antibody surface. Varying concentrations (ranging from 50 nM to 6.25 nM) of protein were injected at 100 μl/min over captured antibody surface for 90 seconds. Antigen-antibody binding and dissociation were monitored in real time at room temperature. Kinetic analysis was performed to calculate K_(D) and half-life of antigen/antibody complex dissociation. The results are summarized in Table 3 below.

TABLE 3 Dimeric Dimeric Human Ang-2FD Mouse Ang-2FD Dimeric K_(D) K_(D) Monkey Ang-2FD Antibody (pM) T_(1/2) (min) (pM) T_(1/2) (min) K_(D) (pM) T_(1/2) (min) H1M724N 179 42.7 694 16 730 25.7 H1M728N 137 58.4 5650 9.9 1580 69.5 H2M730N 210 47 — — 842 36.6 H1M732N 484 35.5 1700 21.4 7330 24.1 H1M737N 251 34.5 1740 6.3 3810 16 H2M742N 295 38 610 30.8 6170 28.5 H2M743N 154 167 882 195.2 234 169.2 H2M744N 98.9 109.1 143 223.1 500 281.7 H2M749N 165 42.9 529 25.5 1500 40.9 H2M750N 362 32.2 — — 1470 23

The above experiment was repeated using selected purified anti-Ang-2 antibodies cloned onto human IgG1. The results are summarized in Table 4 below.

TABLE 4 Dimeric Dimeric Dimeric Human Ang-2FD Mouse Ang-2FD Monkey Ang-2FD Antibody K_(D) (pM) T_(1/2) (min) K_(D) (pM) T_(1/2) (min) K_(D) (pM) T_(1/2) (min) H1H685P 71.4 229.4 148 128.7 99.4 177.1 H1H690P 79 126.1 91.3 105.2 55.6 195.2 H1H691P 220 38.5 220 43.8 290 41 H1H693P 500 37.1 446 63.7 1170 17.6 H1H694P 126 265.6 237 166.5 356 85.6 H1H695P 245 147 347 124.2 440 84.1 H1H696P 289 38.8 402 37.6 354 36.6 H1H704P 331 86.1 484 61.9 818 33.5 H1H706P 201 50.4 357 47 164 53.3 H1H707P 262 26.6 328 34.4 283 22.3 H1H724N 115 107 185 84 239 173 H1H728N 162 81 5760 20 2000 77 H1H730N 234 62 97.1 90 3400 87 H1H732N 386 57 529 51 439 118 H1H742N 186 65 276 58 683 93 H1H743N 88.2 254 124 233 96.5 780 H1H744N 114 127 158 115 346 164 H1H749N 118 109 177 96 407 143 H1H750N 164 127 218 121 199 244 Control I (Ab) 339 34.8 339 47.1 537 27.1

Additional binding experiments were conducted using selected anti-Ang-2 antibodies at two different temperatures to further assess cross-species affinity. Each selected antibody or control construct was captured at a flow rate of 40 μL/min for 1 minute on a goat anti-human kappa polyclonal antibody surface created through direct chemical coupling to a BIACORE™ chip to form a captured antibody surface. Human, monkey and mouse Ang-2FD-Fc at a concentration of 25 nM or 0.78 nM was injected over the captured antibody surface at a flowrate of 60 μL/min for 3 minutes, and antigen-antibody dissociation was monitored in real time for 20 minutes at either 25° C. or 37° C.

Results are summarized in Tables 5 (25° C. binding) and 6 (37° C. binding) below.

TABLE 5 Binding at 25° C. Dimeric Dimeric Dimeric Human Ang-2FD-mFc Mouse Ang-2FD-hFc Monkey Ang-2FD-hFc Antibody K_(D) (Molar) T_(1/2) (min) K_(D) (Molar) T_(1/2) (min) K_(D) (Molar) T_(1/2) (min) H1H685P 1.17E−11 227 6.51E−11 208 2.20E−11 275 H1H744N 1.16E−10 23 3.85E−10 33 2.44E−10 24 Control I (Ab) 1.07E−09 15 1.07E−09 15 1.03E−09 4 Control IV (Ab) 1.27E−11 269 4.02E−11 289 1.55E−11 342

TABLE 6 Binding at 37° C. Dimeric Dimeric Dimeric Human Ang-2FD-mFc Mouse Ang-2FD-hFc Monkey Ang-2FD-hFc Antibody K_(D) (Molar) T_(1/2) (min) K_(D) (Molar) T_(1/2) (min) K_(D) (Molar) T_(1/2) (min) H1H685P 2.70E−11 60 9.39E−11 64 7.21E−11 65 H1H744N 1.05E−10 18 2.15E−10 26 3.20E−10 11 Control I (Ab) — — 3.90E−10 12 — — Control IV (Ab) 9.91E−12 184  5.40E−11 119 4.74E−11 107 

In another experiment, K_(D) values for selected purified antibodies that bind to a human “bow-Ang-2” tetrameric construct (“hBA2”) were determined (using the methods described above). hBA2 consists of two dimers, each dimer containing two Ang-2 fibronectin-like domains connected to one another by a human Fc domain. The amino acid sequence of the dimer constituents of hBA2 is represented by SEQ ID NO:522. The results are summarized in Table 7 below.

TABLE 7 hBA2 Antibody K_(D) (pM) T_(1/2) (min) H1H685P 11.9 587.2 H1H690P 17.9 299.3 H1H691P 106 50.6 H1H693P 299 28.7 H1H694P 68.4 111.3 H1H695P 40.1 254.3 H1H696P 111 51.5 H1H704P 93.9 117.7 H1H706P 79.1 63.9 H1H707P 75.2 51.4 H1H724N 23.3 323 H1H728N 41.8 185 H1H730N 55.9 152 H1H732N 132 73 H1H742N 72.1 87 H1H743N 9.71 1118 H1H744N 17.2 442 H1H749N 32.5 235 H1H750N 36.9 284 Control I (Ab) 83 57.5

In yet another experiment, K_(D) values for selected purified antibodies that bind to wild-type human Ang-2 (hAng-2-WT; SEQ ID NO: 518) and the fibrinogen-like domain of human Ang2 (hAng-2FD) were determined (as described above). The results are summarized in Table 8 below.

TABLE 8 Monomeric hAng-2FD hAng-2-WT Antibody K_(D) (nM) T_(1/2) (min) K_(D) (pM) T_(1/2) (min) H1M724N 1.75 17.4 33.1 568 H1M728N 1.17 33.9 33.8 725 H2M730N 2.06 24.4 49.2 519 H1M732N 6.13 18.7 131 333 H1M737N 2.82 13.1 59.3 282 H2M742N 4.81 18.0 67.9 437 H2M743N 0.399 156.7 14.3 2366 H2M744N 0.475 89.3 28.9 846 H2M749N 1.38 27.9 49 479 H2M750N 4.42 21.5 40.8 991 H1H685P 0.578 55 47.6 1000 H1H691P 11 0.57 19.1 684.6 H1H690P 0.594 25.16 12.4 1568 H1H693P 44.8 0.61 425 100 H1H694P 7.89 9.85 158 209.7 H1H695P 1.12 50.59 31.1 1770.7 H1H696P 38.4 0.20 40.3 642.7 H1H704P 0.39 3.31 36.2 747.6 H1H706P 11 1.02 27.4 661.9 H1H707P 145 — 77.1 217.4 H1H724N 2.4 13.34 22.6 895 H1H728N 1.18 5.86 43 566 H1H730N 2.84 3.44 47.5 534 H1H732N 264 0.22 202 264 H1H742N 486 2.29 44.9 666 H1H743N 2.35 33.03 9.48 3927 H1H744N 1.02 42.14 30.8 837 H1H749N 1.13 33.48 12.5 1833 H1H750N 0.787 30.20 9.5 4442 Control I (Ab) 44.5 0.03 47.6 512 Control II (Pb) 90 — 44.7 334.8

Additional experiments were conducted to measure the binding properties of selected anti-Ang-2 antibodies to monomeric hAng-2FD at 25° C. and 37° C. Each selected antibody or control construct was captured at a flow rate of 40 μL/min for 1 minute on a goat anti-human IgG polyclonal antibody surface created through direct chemical coupling to a BIACORE™ chip to form a captured antibody surface. Human Ang-2FD at a concentration of 500 nM or 7.8 nM was injected over the captured antibody surface at a flowrate of 60 μL/min for 3 minutes, and antigen-antibody dissociation was monitored in real time for 20 minutes at either 25° C. or 37° C.

Results are summarized in Tables 9 (25° C.) and 10 (37° C.) below. N/D=not determined.

TABLE 9 Binding to monomeric hAng-2FD at 25° C. K_(D) ka (Ms⁻¹) kd (s⁻¹) (Molar) T½ H1H685P 2.44E+05 7.96E−05 3.36E−10 145 minutes H1H744N 2.92E+05 1.24E−04 4.24E−10 93 minutes Control I (Ab) 4.00E+05 5.10E−02 1.28E−07 14 seconds Control II (Pb) steady-state steady-state 9.00E−08 steady-state Control III (Pb) 5.40E+05 6.30E−02 1.17E−07 11 seconds Control IV 2.84E+05 3.56E−02 1.25E−07 19 seconds (Ab)

TABLE 10 Binding to monomeric hAng-2FD at 37° C. K_(D) ka (Ms⁻¹) kd (s⁻¹) (Molar) T½ H1H685P 4.06E+05 1.39E−04 3.42E−10 83 minutes H1H744N 3.86E+05 5.48E−04 1.42E−09 21 minutes Control I (Ab) steady-state steady-state 1.51E−07 steady-state Control II (Pb) N/D N/D N/D N/D Control III (Pb) steady-state steady-state 2.94E−07 steady-state Control IV (Ab) steady-state steady-state 9.40E−08 steady-state

As shown in this Example, several of the anti-Ang-2 antibodies generated in accordance with the methods of Example 1 bound to Ang-2 constructs with equivalent or higher affinities than the controls. For example, antibodies H1H685, H1H690, H1H724 and H1H744 bound to dimeric human Ang-2-FD with K_(D)'s of 71.4, 79, 115, and 114 pM, respectively, whereas Control I antibody bound to dimeric human Ang-2-FD with a K_(D) of 339 pM (see Table 4). Similarly, antibodies H1H685, H1H690, H1H724 and H1H744 bound to human BA2 (a tetrameric Ang-2 fibrinogen-like domain construct) with K_(D)'s of 11.9, 17.9, 23.3 and 17.2 pM, respectively, whereas Control I antibody bound to hBA2 with a K_(D) of 83 pM (see Table 7). Thus, as compared to the control constructs, many of the antibodies of the invention exhibit enhanced binding to Ang-2. Antibody H1H685P showed especially robust binding properties to Ang-2 as compared to the control constructs.

Example 4. Preferential Binding to Ang-2 Over Ang-1

Binding experiments (plasmon resonance assays) were conducted to ascertain whether selected antibodies bound to both Ang-2 and Ang-1 or if they preferentially bound to Ang-2 only. Each selected antibody or control construct was captured at a flow rate of 40 μL/min for 1 minute on a goat anti-human IgG polyclonal antibody surface created through direct chemical coupling to a BIACORE™ chip to form a captured antibody surface. Full-length wild-type human Ang-1 or Ang-2 at a concentration of 25 nM or 0.78 nM were injected over the captured antibody surface at a flowrate of 60 μL/min for 3 minutes, and antigen-antibody dissociation was monitored in real time for 20 minutes at either 25° C. or 37° C.

The results of these experiments are summarized in Tables 11-14 below. N/D=not determined. “No binding” means that no detectable binding was observed under the particular experimental conditions used in these experiments.

TABLE 11a Binding to hAng-2-WT at 25° C. K_(D) T½ ka (Ms⁻¹) kd (s⁻¹) (Molar) (minutes) H1H685P 6.59E+05 1.60E−05 2.42E−11 722 H1H744N 7.65E+05 2.57E−05 3.35E−11 450 Control I (Ab) 4.74E+05 2.26E−05 4.76E−11 512 Control II (Pb) 7.73E+05 3.45E−05 4.47E−11 335 Control III (Pb) 3.29E+05 1.98E−05 6.01E−11 584 Control IV (Ab) 3.80E+06 2.74E−04 7.22E−11 42

TABLE 11b Binding to hAng-2-WT at 25° C. K_(D) T½ ka (Ms⁻¹) kd (s⁻¹) (Molar) (minutes) H1H685P 1.15E+05 8.50E−06 7.39E−11 1359 Control II (Pb) 8.30E+04 5.41E−05 6.52E−10 213 Control V (Ab) 1.12E+05 2.66E−05 2.73E−10 434

TABLE 12a Binding to hAng-1-WT at 25° C. K_(D) T½ ka (Ms⁻¹) kd (s⁻¹) (Molar) (minutes) H1H685P No binding No binding No binding No binding H1H744N 4.10E+05 3.81E−05 9.30E−11 303 Control I (Ab) 4.55E+05 2.49E−05 5.47E−11 464 Control II (Pb) 4.53E+05 3.54E−05 7.82E−11 326 Control III (Pb) No binding No binding No binding No binding Control IV 6.60E+05 1.11E−04 1.68E−10 105 (Ab)

TABLE 12b Binding to hAng-1-WT at 25° C. K_(D) T½ ka (Ms⁻¹) kd (s⁻¹) (Molar) (minutes) H1H685P No binding No binding No binding No binding Control II (Pb) 3.04E+05 2.51E−05 8.26E−11 460 Control V (Ab) 2.75E+05 6.68E−05 2.43E−10 173

TABLE 13a Binding to hAng-2-WT at 37° C. K_(D) T½ ka (Ms⁻¹) kd (s⁻¹) (Molar) (minutes) H1H685P 8.54E+05 3.76E−05 4.40E−11 707 H1H744N 7.01E+05 2.43E−04 3.47E−10 48

TABLE 13b Binding to hAng-2-WT at 37° C. K_(D) T½ ka (Ms⁻¹) kd (s⁻¹) (Molar) (minutes) H1H685P 1.36E+05 2.16E−05 1.59E−10 535 Control II (Pb) 3.79E+04 1.17E−04 3.09E−09 99 Control V (Ab) 9.42E+04 7.92E−05 8.41E−10 146

TABLE 14a Binding to hAng-1-WT at 37° C. K_(D) T½ ka (Ms⁻¹) kd (s⁻¹) (Molar) (minutes) H1H685P No binding No binding No binding No binding H1H744N 1.47E+06 5.20E−05 3.12E−11 222 Control III (Pb) No binding No binding No binding No binding

TABLE 14b Binding to hAng-1-WT at 37° C. K_(D) T½ ka (Ms⁻¹) kd (s⁻¹) (Molar) (minutes) H1H685P No binding No binding No binding No binding Control II (Pb) 2.81E+05 4.35E−05 1.55E−10 266 Control V (Ab) 4.42E+05 5.47E−05 1.24E−10 211

These results show that H1H685P is unique among the antibodies tested in this experiment in that it binds with high affinity to Ang-2 but does not bind to Ang-1. The only other construct that exhibits binding to Ang-2 but not to Ang-1 is Control III. It should be emphasized, however, that Control III is a peptibody and that all of the other antibodies tested in this experiment bound to both Ang-2 and Ang-1. The selectivity for Ang-2 binding may confer therapeutic benefits on H1H685P that are not possessed by antibodies that bind to both Ang-2 and Ang-1.

Example 5. Inhibition of Ang-2 Binding to Human Tie-2

Tie-2 is a natural receptor for Ang-2. Anti-Ang-2 antibodies were tested for their ability to block Ang-2 binding to human Tie-2 (hTie-2). hTie-2-mFc (a chimeric construct consisting of human Tie-2 conjugated to mouse IgG; SEQ ID NO:525) was coated onto 96-well plates at a concentration of 2 μg/ml and incubated overnight followed by washing four times in wash buffer (PBS with 0.05% Tween-20). The plate was then blocked with PBS (Irvine Scientific, Santa Ana, Calif.) containing 0.5% BSA (Sigma-Aldrich Corp., St. Louis, Mo.) for one hour at room temperature. In a separate plate, purified anti-Ang-2 antibodies, at a starting concentration of 50 nM, were serially diluted by a factor of three across the plate. Human, mouse or monkey Ang-2FD protein conjugated to human IgG (Ang-2FD-hFc) were added to final concentrations of 2 nM, 8 nM, or 2 nM respectively and incubated for one hour at room temperature. The antibody/Ang-2FD-Fc mixture was then added to the plate containing hTie-2-mFc and incubated for one hour at room temperature. Detection of Ang-2FD-hFc bound to hTie-2-mFc protein was determined with Horse-Radish Peroxidase (HRP) conjugated to a-human IgG antibody (Jackson Immuno Research Lab, West Grove, Pa.) and developed by standard colorimetric response using tetramethylbenzidine (TMB) substrate (BD Biosciences, San Jose, Calif.). Absorbance was read at OD₄₅₀ for 0.1 sec. Percent blocking of Ang-2FD-hFc binding to hTie-2-mFc by 16.67 nM of selected anti-Ang-2 antibodies is shown in Table 15.

TABLE 15 Percent Blocking of Ang-2FD Binding to Tie-2 Human Mouse Monkey Antibody Ang-2FD-hFc Ang-2FD-hFc Ang-2FD-hFc H1M724N 99.5 96.6 95.2 H1M728N 98.5 83.9 97.1 H2M730N 98.9 55.0 97.3 H1M732N 97.7 90.9 95.8 H1M737N 99.1 95.4 90.5 H2M742N 99.6 98.6 94.1 H2M743N 99.6 98.4 95.1 H2M744N 99.5 98.4 95.5 H2M749N 99.5 97.3 97.4 H2M750N 99.4 53.7 97.4 Control I (Ab) 94.5 90.2 96.9

In a similar experiment, selected purified anti-Ang-2 antibodies cloned onto human IgG1 were tested for their ability to block Ang-2FD binding to hTie-2 (as described above). Percent blocking of Ang-2FD-hFc binding to hTie-2-mFc by 16.67 nM of selected anti-Ang-2 antibodies is shown in Table 16. NT: not tested.

TABLE 16 Percent Blocking of Ang-2FD Binding to Tie-2 Human Mouse Monkey Antibody Ang-2FD-hFc Ang-2FD-hFc Ang-2FD-hFc H1H685P 93.8 97.1 62.2 H1H690P 97.2 98.0 99.6 H1H691P 97.4 96.7 99.8 H1H693P 73.9 63.6 NT H1H694P 79.8 36.0 NT H1H695P 98.4 97.6 NT H1H696P 98.2 94.9 99.2 H1H704P 97.0 41.8 NT H1H706P 97.1 95.9 99.8 H1H707P 95.1 93.8 NT H1H724N 96.6 97.1 96.6 H1H744N 97.9 97.6 96.2 Control I (Ab) 97.3 82.2 98.5

In another experiment, selected purified anti-Ang-2 antibodies were tested for their ability to block binding of 20 pM biotinylated hBA2 to hTie-2 (as described above). For this experiment, human Tie-2 conjugated to a histidine tag (hTie-2-His; SEQ ID NO:526) was used in a similar fashion to the hTie-2-mFc described above. Antibody concentrations from 5 nM were serially diluted three-fold. An IC₅₀ (Inhibitory Concentration) value was generated by calculating the amount of antibody required to block 50% of the signal from the binding of biotin-hBA2 to Tie-2. An average IC₅₀ value for each antibody was calculated based on two separate experiments. The results are summarized in Table 17. NB: no blocking observed at 5 nM concentration.

TABLE 17 Biotin - hBA2 Antibody Average IC₅₀ (pM) H1M724N 9.72 H1M728N 14.05 H2M730N 14.60 H1M732N 82.17 H1M737N 13.01 H2M742N 9.65 H2M743N 11.01 H2M744N 11.43 H2M749N 6.43 H2M750N 8.83 Control I (Ab) 30.23 Control II (Pb) 7.75 Control III (Pb) 16.49

In a similar experiment, selected purified anti-Ang-2 antibodies cloned onto human IgG1 were tested for their ability to block binding of biotinylated hBA2 to hTie-2 (as described above). The results are shown in Table 18. NB: no blocking observed at 5 nM concentration.

TABLE 18 Biotin - hBA2 Antibody IC₅₀ (pM) H1H685P 20 H1H690P 17 H1H691P 13 H1H693P NB H1H694P NB H1H695P 59 H1H696P 22 H1H704P 56 H1H706P  8 H1H707P 22 H1H724N  4 H1H744N 25

This Example illustrates that several of the anti-Ang-2 antibodies generated in accordance with the methods of Example 1 blocked the interaction between the Ang-2 fibrinogen-like domain and its receptor (TIE-2) to an equivalent or greater extent than the control antibody. For example, antibodies H1H690, H1H691, H1H695, H1H696, H1H704, H1H706, H1H707, H1H724 and H1H744 each caused greater than 95% blocking of human, mouse and monkey Ang-2FD constructs to the TIE-2 receptor, similar to the results observed with the control constructs (see Table 16).

Example 6. Inhibition of Full-Length Ang-2 and Ang-1 Binding to Human Tie-2

Tie-2 is a receptor for Ang-1 as well as Ang-2. Therefore, in the present Example, the ability of certain anti-Ang-2 antibodies to block binding of Ang-2 or Ang-1 to human Tie-2 was measured and compared.

The ELISA experiments shown in this Example were conducted in a similar manner to the experiments of Example 5. Briefly, hTie-2-mFc (a chimeric construct consisting of human Tie-2 conjugated to mouse IgG; SEQ ID NO:525) was coated onto 96-well plates at a concentration of 2 μg/ml and incubated overnight followed by washing four times in wash buffer (PBS with 0.05% Tween-20). The plate was then blocked with PBS (Irvine Scientific, Santa Ana, Calif.) containing 0.5% BSA (Sigma-Aldrich Corp., St. Louis, Mo.) for one hour at room temperature. In a separate plate, purified anti-Ang-2 antibodies and control constructs, at a starting concentration of 300 nM, were serially diluted by a factor of three across the plate. Full-length human Ang-2 or Ang-1 protein conjugated to 6× histidine tag (R&D Systems, Minneapolis, Minn.) were added to a final concentration of 0.6 nM and incubated for one hour at room temperature. The antibody/antigen mixture was then added to the plate containing hTie-2-mFc and incubated for one hour at room temperature. Detection of Ang-2-His or Ang-1-His bound to hTie-2-mFc protein was determined with Horse-Radish Peroxidase (HRP) conjugated to a-Penta-His antibody (Qiagen, Valencia, Calif.) and developed by standard colorimetric response using tetramethylbenzidine (TMB) substrate (BD Biosciences, San Jose, Calif.). Absorbance was read at OD₄₅₀ for 0.1 sec. An IC₅₀ (Inhibitory Concentration) value was generated by calculating the amount of antibody required to block 50% of the signal from the binding of human Ang-2 or Ang-1 to Tie-2. The results, expressed in terms of IC₅₀ are shown in Table 19, columns (1) and (2). The extent to which the antibodies or control constructs block the hAng-2/Tie-2 interaction relative to the hAng-1/Tie-2 interaction is reflected in the fold difference in IC₅₀ shown in column (3); that is, a higher number in column (3) indicates a greater capacity to block the hAng-2/Tie-2 interaction than the hAng-1/Tie-2 interaction.

TABLE 19 (3) Fold Difference in (1) Blocking (2) Blocking hAng-1 Blocking hAng-2 WT hAng-1 WT IC₅₀ Compared to Tie-2 to Tie-2 to h-Ang-2 Antibody IC₅₀ (M) IC₅₀ (M) Blocking IC₅₀* H1H685P 1.294E−10 >3.000E−07  >2318 H1H744N 7.871E−11 1.872E−07 2378 Control I (Ab) 9.372E−11 6.171E−08 658 Control II (Pb) 3.096E−11 5.509E−11 1.8 Control III (Pb) 1.626E−10 >1.000E−06  >6150 Control IV (Ab) 1.476E−10 4.252E−09 28.8 *Calculated by dividing the hAng-1 blocking IC₅₀ (column 2) by the hAng-2 blocking IC₅₀ (column 1)

In an effort to further assess the ability of selected anti-hAng-2 antibodies to block the binding of Ang-1 to Tie-2, a biosensor surface plasmon resonance experiment was conducted. In this experiment, a human Tie-2 full-length extracellular domain construct (hTie-2-mFc-ecto) was amine-coupled on a BIACORE™ chip to create a receptor coated surface. Selected anti-hAng-2 antibodies and control constructs, at 1 μM (100-fold excess over antigen), were premixed with 10 nM of hAng-1-WT, followed by 60 minutes incubation at 25° C. to allow antibody-antigen binding to reach equilibrium to form equilibrated solutions. The equilibrated solutions were injected over the receptor surfaces at 5 μL/min for 5 minutes at 25° C. Changes in resonance units (RU) due to the binding of the hAng-1-WT to hTie-2-mFc were determined. An irrelevant peptibody construct with no binding to hAng-1 was included in this experiment to establish the 0% blocking baseline, and a human Tie-2-mFc construct was used as a positive control for blocking. The amount of Ang-1 bound to Tie-2 following antibody preincubation, expressed as a percentage of the amount of Ang-1 bound to Tie-2 following negative control preincubation, is shown in Table 20. (A greater amount of Ang-1 binding to Tie-2 signifies a lower degree of antibody blocking).

TABLE 20 Percent of Negative Control Antibody RU (average) Binding Negative Control 169 100 (irrelevant peptibody) hTie-2-mFc 71 42 H1H685P 137 81 H1H744N 57 34 H1H691P 117 69 H1H706P 140 83 H1H724N 57 34 Control I (Ab) 48 28 Control II (Pb) 48 28 Control III (Pb) 160 95

The foregoing experiment was repeated using different amounts of Ang-2 blockers and controls. In particular, a human Tie-2 full-length extracellular domain construct (hTie-2-mFc-ecto) was amine-coupled on a BIACORE™ chip to create a receptor-coated surface. Selected anti-hAng-2 antibodies and control constructs (50 or 150 nM) were mixed with hAng-2-WT (25 nM) followed by 60 minutes incubation at 25° C. to allow antibody-antigen binding to reach equilibrium. The equilibrated solutions were injected over the receptor surfaces at 10 μL/min for 5 minutes at 25° C. To evaluate the ability of the selected anti-hAng-2 antibodies to block Ang-1-WT binding to hTie-2, a similar procedure was followed except the antibodies were tested at three concentrations (50, 100 or 1000 nM) and incubated with 10 nM of hAng-1-WT. Changes in resonance units (RU) due to the binding of the Ang-2-WT or hAng-1-WT to hTie-2-mFc were determined. An irrelevant antibody with no binding to either angiopoietin was included in these experiments to establish the 0% blocking baseline, and a human Tie-2-mFc construct was used as a positive control for blocking. Results are summarized in Tables 21 (hAng-1 applied to a hTie-2 surface) and 22 (hAng-2 applied to a hTie-2 surface).

TABLE 21 (hAng-1 WT) Amount of Antibody or Control 50 nM 100 nM 1000 nM Percent of Percent of Percent of Specific Neg. Ctrl Specific Neg. Ctrl Specific Neg. Ctrl Antibody Bound RU Binding Bound RU Binding Bound RU Binding Negative Control 316 100 307 100 276 100 (irrelevant antibody) hTie-2-mFc 70 22 39 13 −47 0 H1H685P 299 95 291 95 289 105 Control II (Pb) 8 2.5 4 1.3 −1 0 Control V (Ab) 150 48 114 37 29 11

TABLE 22 (hAng-2 WT) Amount of Antibody or Control 50 nM 150 nM Percent of Percent of Specific Neg. Ctrl Specific Neg. Ctrl Antibody Bound RU Binding Bound RU Binding Negative Control 281 100 278 100 (irrelevant antibody) hTie-2-mFc 97 35 82 30 H1H685P 12 4.3 12 4.3 Control II (Pb) 10 3.6 10 3.6 Control V (Ab) 12 4.3 12 4.3

The results obtained from these experiments are in agreement with previous results which showed that H1H685P preferentially binds to Ang-2 over Ang-1 (see Example 4). In particular, the results from this Example show that several anti-Ang-2 antibodies (e.g., H1H685P and H1H706P) do not significantly block the binding of human Ang-1 to human Tie-2, even though, in other experiments, it was demonstrated that these antibodies potently blocked the interaction between Ang-2 and Tie-2 (see Example 5, Table 16). Moreover, in these experiments none of the control constructs, except for the Control III peptibody, exhibited the same degree of preferential binding/blocking of Ang-2 over Ang-1 as the exemplary anti-Ang-2 antibodies of the present invention, such as H1H685P.

Example 7. Inhibition of Ang-2-Mediated Tie-2 Phosphorylation by Anti-Ang-2 Antibodies

The inventors of the present invention have demonstrated that Ang-2 expression can be induced in human umbilical vein endothelial cells (HUVECs) by the transcription factor FOXO1 (Daly et al. 2006 PNAS 103:15491). Further, the inventors have shown that infection of HUVECs with an adenovirus encoding FOXO1 results in expression and secretion of Ang-2, followed by activation of Tie-2 phosphorylation (Daly et al. 2006 PNAS 103:15491).

Anti-Ang-2 antibodies were tested for their ability to inhibit Tie-2 phosphorylation. Briefly, 7×10⁵ HUVECs (Vec Technologies, Rensselaer, N.Y.) were plated in 6 cm cell culture dishes in 3.5 ml of MCDB131 Complete medium (Vec Technologies, Rensselaer, N.Y.). The following day, the cells were washed with Opti-MEM (Invitrogen Corp., Carlsbad, Calif.) and fed with 2 ml of Opti-MEM. Recombinant adenoviruses encoding either green fluorescent protein (GFP; control) or human FOXO1 (Daly et al. 2004 Genes Dev. 18:1060) were added to the cells at a concentration of 10 pfu/cell and incubated for four hours. Cells were then washed with MCDB131 and fed with 2 ml of MCDB131 containing anti-Ang-2 antibodies at a concentration of 0.5 μg/ml. At twenty hours post infection, cells were lysed and subjected to Tie-2 immunoprecipitation as described by Daly et al., Proc. Natl. Acad. Sci. USA 103:15491-15496 (2006). Immunoglobulin was collected on protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, Calif.) for one hour. Beads were washed with cold lysis buffer and resuspended in SDS sample buffer for analysis by western blot with antibodies specific for phosphotyrosine (Millipore, Billerica, Mass.) or Tie-2. Signals were detected using HRP-conjugated secondary antibodies and ECL reagents (GE Healthcare, Piscataway, N.J.). X-Ray films were scanned and the phospho-Tie-2 and Tie-2 signals were quantified using ImageJ software. The phospho-Tie-2/Tie-2 ratios were used to determine the % inhibition for each anti-Ang-2 antibody (i.e. Percent inhibition=Reduction in phospho-Tie-2/Tie-2 as compared to control). For example, a reduction in Tie-2 phosphorylation to the level observed in the control sample is considered to be 100% inhibition. Relative inhibition (+, ++, +++) for each anti-Ang-2 antibody tested according to the percent inhibition observed (25-50%, 50-75%, 75-100%, respectively) is shown in Table 23.

TABLE 23 Inhibition of Antibody Tie-2 phosphorylation H1H685P +++ H1H690P +++ H1H691P +++ H1H693P +++ H1H694P ++ H1H695P +++ H1H696P +++ H1H704P +++ H1H706P +++ H1H707P +++ H1M724N +++ H1M728N ++ H1M732N ++ H1M742N ++ H1M743N +++ H1M744N +++ H1M749N ++ H1M750N +++ Control I (Ab) + Control II (Pb) +++

As demonstrated in this Example, the anti-Ang-2 antibodies generated in accordance with the methods of Example 1 inhibited Tie-2 phosphorylation to a greater extent than the Control I antibody. Especially robust inhibition was observed with antibodies H1H685, H1H690, H1H691, H1H693, H1H695, H1H696, H1H704, H1H706, H1H707, H1M724, H1M744 and H1M750.

Example 8. Inhibition of Ang-1-Mediated Tie-2 Phosphorylation

As shown in the previous Example, Ang-2 can mediate the phosphorylation of Tie-2. Ang-1 is also capable of promoting Tie-2 phosphorylation. In the present Example, the ability of selected anti-Ang-2 antibodies to block Ang-1-mediated phosphorylation of Tie-2 was assessed.

EA.hy926 cells (Edgell et al., Proc. Natl. Acad. Sci. USA 80:3734-3737 (1983)) were plated at 5×10⁶ cells per 10 cm dish in 10 ml DMEM with 10% FBS, HAT, L-glutamine and penicillin/streptomycin. After 24 hours, cells were serum-starved for 1 hour in 10 ml DMEM+1 mg/ml BSA. Cells were then stimulated for 10 minutes with 500 ng/ml of recombinant human Ang-1 (R&D Systems) in the presence of either an irrelevant isotype control antibody (“9E10”) at 400 nM or the anti-Ang-2 antibody H1H685P, or control agents (Control I, Control II, Control IV, or Control V) at concentrations ranging from 10 to 400 nM.

Following incubation, cells were lysed and Tie-2 was immunoprecipitated as described by Daly et al., Proc. Natl. Acad. Sci. USA 103:15491-15496 (2006). Immune complexes were collected by incubation with protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, Calif.) for 60 min. Beads were washed with cold lysis buffer and bound proteins were eluted by heating in SDS sample buffer. Samples were then subjected to Western blot analysis with monoclonal antibodies against Tie-2 or phosphotyrosine (clone 4G10, Millipore, Billerica, Mass.). Results are shown in FIG. 2.

Signals were detected using HRP-conjugated secondary antibodies and ECL reagents (GE Healthcare, Piscataway, N.J.). X-ray films were scanned and the phospho-Tie-2 and Tie-2 signals were quantified using ImageJ software. The phospho-Tie-2/Tie-2 ratios were used to determine the % inhibition for each antibody or peptibody. Percent inhibition=reduction in phospho-Tie-2/Tie-2 as compared to the control sample (400 nM isotype control antibody).

In the presence of the control antibody 9E10, Ang-1 strongly activated Tie-2 phosphorylation (FIG. 2, panel A—compare lanes 2 and 3 vs lane 1). All of the control agents that were tested significantly inhibited Tie-2 phosphorylation, with complete inhibition occurring at 50 nM for Control II (FIG. 2, panel B—lane 17), 100 nM for Control IV (FIG. 2, panel A—lane 11) and 200 nM for Control I (FIG. 2, panel B—lane 24) and Control V (FIG. 2, panel C—lane 9). By contrast, H1H685P had no significant inhibitory effect even at 400 nM (FIG. 2, panel A—lanes 4-8). These results provide additional confirmation of the specificity of H1H685P for Ang-2 over Ang-1.

Example 9. Inhibition of Tumor Growth by Anti-Ang-2 Antibodies

The effect of selected purified anti-Ang-2 antibodies on tumor growth was determined using two tumor cell lines.

PC3 (Human Prostate Cancer Cell Line)

Briefly, 5×10⁶ PC3 cells in 100 μl of growth factor-reduced Matrigel (BD Biosciences) were injected subcutaneously into the flanks of 6-8 week old male NCr nude mice (Taconic, Hudson, N.Y.). After tumor volumes reached an average of about 200 mm³, mice were randomized into groups for treatment. Mice in each treatment group were administered an anti-Ang-2 antibody, Fc protein, or control construct, at a concentration of 10 mg/kg via intraperitoneal injection twice per week for approximately three weeks (Table 24) or at concentrations of 2.5, 12.5, or 25 mg/kg via subcutaneous injection twice per week for approximately three weeks (Table 25). Tumor volumes were measured twice per week over the course of the experiment and tumor weights were measured upon excision of tumors at the conclusion of the experiment. Averages (mean+/−standard deviation) of tumor weight and growth were calculated for each treatment group. Percent decrease of tumor weight and growth were calculated from comparison to Fc protein measurements. Results are summarized in Tables 24 and 25.

TABLE 24 Avg Tumor Avg Tumor % Decrease in Growth % Decrease in Antibody Weight (g) Tumor Weight (mm³) Tumor Growth Fc protein 0.66 ± 0.26 — 509 ± 213 — Control I 0.47 ± 0.23 29 300 ± 242 41 (Ab) H1H724N 0.55 ± 0.07 17 392 ± 169 23 H1H744N 0.43 ± 0.20 35 259 ± 212 49 H1H685P 0.44 ± 0.12 33 305 ± 143 40 H1H691P 0.59 ± 0.07 11 485 ± 141  5 H1H706P 0.52 ± 0.14 21 329 ± 125 35

TABLE 25 Avg Tumor % Decrease in Antibody Growth (mm³) Tumor Growth Fc protein 1031 ± 485  — Control II (Pb) 356 ± 196 65 (2.5 mg/kg) Control II (Pb) 360 ± 162 65 (12.5 mg/kg) Control II (Pb) 527 ± 218 49 (25 mg/kg) H1H685P 308 ± 274 70 (2.5 mg/kg) H1H685P 550 ± 150 47 (12.5 mg/kg) H1H685P 413 ± 208 60 (25 mg/kg)

As shown above, antibodies H1H744N and H1H685P demonstrated especially marked anti-tumor activity in the PC3 mouse tumor model as compared to the control constructs.

The results of similar experiments using the PC3 mouse tumor model and different experimental antibodies (dosed at 2 mg/kg, twice per week) are shown in Tables 26 and 27.

TABLE 26 % Avg Decrease Tumor in Avg Tumor % Decrease in Growth Tumor Antibody Weight (g) Tumor Weight (mm³) Growth Fc protein 0.626 ± 0.156 — 356 ± 93  — Control I (Ab) 0.347 ± 0.093 45 250 ± 145 30 H2M742N 0.407 ± 0.076 35 220 ± 102 38 H2M743N 0.372 ± 0.122 41 179 ± 169 50

TABLE 27 % Decrease % Decrease Avg Tumor in Tumor Avg Tumor in Tumor Antibody Weight (g) Weight Growth (mm³) Growth Fc protein 0.552 ± 0.211 — 473 ± 202 — H1M749N 0.383 ± 0.275 31 220 ± 261 54 H1M750N 0.348 ± 0.128 37 227 ± 195 52

COLO 205 (Human Colorectal Adenocarcinoma Cell Line)

Briefly, 2×10⁶COLO 205 cells in 100 μl of serum-free medium were injected subcutaneously into the flank of 6-8 week old male NCr nude mice (Taconic, Hudson, N.Y.). After tumor volumes reached an average of about 150 mm³, mice were randomized into groups for treatment with antibody or Fc protein. Mice in each treatment group were administered an anti-Ang-2 antibody or Fc protein at a concentration of 4 mg/kg via intraperitoneal injection twice per week for approximately two weeks. Tumor volumes were measured twice per week over the course of the experiment and tumor weights were measured upon excision of tumors at the conclusion of the experiment. Averages (mean+/−standard deviation) of tumor weight and growth were calculated for each treatment group. “Avg. Tumor Growth” represents the average growth from the time of treatment initiation (when tumors were approximately 150 mm³). Percent decrease of tumor weight and growth are calculated from comparison to Fc protein measurements. Results are summarized in Table 28.

TABLE 28 % Avg Decrease Tumor in Avg Tumor % Decrease in Growth Tumor Antibody Weight (g) Tumor Weight (mm³) Growth Fc protein 0.847 ± 0.180 — 731 ± 249 — Control I (Ab) 0.503 ± 0.090 41 367 ± 121 50 Control II (Pb) 0.608 ± 0.085 28 492 ± 82  33 H1M724N 0.531 ± 0.103 37 336 ± 125 54 H2M742N 0.576 ± 0.057 32 427 ± 92  42 H2M744N 0.491 ± 0.051 42 409 ± 162 44 H1M749N 0.603 ± 0.142 29 449 ± 169 39

A similar experiment was carried out to assess the effect of H1H685P, in particular, on COLO 205 tumor growth. Briefly, 2×10⁶ COLO 205 cells in 100 μl of serum-free medium were implanted subcutaneously into the right hind flank of 9-11 week-old male SCID CB17 mice. When the tumors reached ˜125 mm³, mice were randomized into 5 groups (n=7-8 mice/group) and treated twice per week with Fc protein (15 mg/kg), H1H685P (5 or 25 mg/kg) or Control II (5 or 25 mg/kg) for a period of 19 days. Tumor volumes were measured twice per week over the course of the experiment and tumor weights were measured upon excision of tumors at the end of the experiment. Averages of tumor weight and growth from the beginning of treatment were calculated for each group. Percent decrease of tumor weight and growth are calculated from comparison to the Fc control group. The results are shown in Table 29.

TABLE 29 Antibody Avg Tumor % Decrease in Avg Tumor % Decrease in Antibody Concentration Weight (g) Tumor Weight Growth (mm³) Tumor Growth Fc protein 25 mg/kg 0.800 ± 0.108 — 675 ± 93  — Control II (Pb)  5 mg/kg 0.481 ± 0.091 40 288 ± 85  57 Control II (Pb) 25 mg/kg 0.393 ± 0.136 51 267 ± 155 60 H1H685P  5 mg/kg 0.458 ± 0.125 43 370 ± 114 45 H1H685P 25 mg/kg 0.430 ± 0.139 46 295 ± 160 56

As with the PC3 mouse tumor model, several of the antibodies of the invention, including H1H685P, exhibited substantial anti-tumor activities in the COLO 205 mouse model that were at least equivalent to the anti-tumor activities exhibited by the control molecules.

Example 10. Inhibition of Tumor Growth and Perfusion by a Combination of an Anti-Ang-2 Antibody and a VEGF Inhibitor

To determine the effect of combining an anti-Ang-2 antibody with a VEGF inhibitor on the growth of COLO 205 xenografts, 2×10⁶ cells were implanted subcutaneously into the right hind flank of 6-8 week-old female SCID mice. When the tumors reached an average volume of ˜350 mm³, mice were randomized into 4 groups (n=6 mice/group) and treated with: human Fc protein (7.5 mg/kg), H1H685P (5 mg/kg), VEGF Trap (see U.S. Pat. No. 7,087,411) (2.5 mg/kg) or the combination of H1H685P+VEGF Trap. Mice were given a total of 3 doses over 10 days of treatment. Tumor volumes were measured twice per week over the course of the experiment. Averages of tumor growth from the start of treatment (mean+/−standard deviation) were calculated for each treatment group. Percent decrease of tumor growth was calculated from comparison to the Fc control group. The results are shown in Table 30. Note that in the VEGF Trap and in the H1H685P+VEGF Trap groups the average tumor size was smaller at the end of treatment than at the beginning, i.e., tumor regression was observed.

TABLE 30 Avg Tumor % Decrease in Antibody Growth (mm³) Tumor Growth Fc protein 366 ± 65 — H1H685P  74 ± 77  80 VEGF Trap −62 ± 44 117 H1H685P + −221 ± 131 160 VEGF Trap

The results of this experiment demonstrate that the combination of H1H685P+VEGF Trap causes a decrease in tumor growth that is greater than the percent decrease in tumor growth caused by either component alone.

To provide additional evidence of combination efficacy, the effect of the H1H685P+VEGF Trap combination on the growth of MMT tumors was assessed. 0.5×10⁶ MMT cells were implanted subcutaneously into the right hind flank of 6-8 week-old female SCID mice. When the tumors reached an average volume of ˜400 mm³, mice were randomized into 4 groups (n=11 mice/group) and treated with: human Fc protein (17.5 mg/kg), H1H685P (12.5 mg/kg), VEGF Trap (5 mg/kg) or the combination of H1H685P+VEGF Trap. The Fc and H1H685P groups were given 3 doses over 9 days. The VEGF Trap and combination groups were given 4 doses over 12 days. Tumor volumes were measured twice per week over the course of the experiment and tumor weights were measured upon excision of tumors at the end of the experiment (due to their large size, tumors from the Fc and H1H685P groups were collected 3 days before tumors from the VEGF Trap and combination groups). Averages (mean+/−standard deviation) of tumor growth from the beginning of treatment and of tumor weight were calculated for each group. Percent decrease of tumor weight and growth are calculated from comparison to the Fc control group. The results are shown in Table 31.

TABLE 31 % Decrease Avg Tumor % Decrease Avg Tumor in Tumor Growth in Tumor Antibody Weight (g) Weight (mm³) Growth Fc protein 1.591 ± 0.265 — 1337 ± 273 — H1H685P 1.409 ± 0.314 11 1135 ± 306 15 VEGF Trap 0.889 ± 0.141 44  536 ± 179 60 H1H685P + 0.599 ± 0.066 62 215 ± 92 84 VEGF Trap

These results confirm the enhanced tumor inhibiting effect of H1H685P+VEGF Trap relative to the single agent treatments.

To determine whether the combination of H1H685P+VEGF Trap has a greater effect on tumor vessel function than the single agents, a micro-ultrasound (VisualSonics' Vevo 770 imaging system) was used to assess changes in tumor perfusion. COLO 205 tumors were grown to ˜125 mm³ and mice were then treated for 24 hrs with H1H685P, VEGF Trap or the combination of both agents. Following treatment, tumor vessel perfusion was determined based on contrast-enhanced micro-ultrasound 2D image acquisition and analysis of a “wash-in” curve, which represents the amount of contrast agent entering the tumor. Average (mean+/−standard deviation) tumor perfusion was calculated for each group. Percent decrease was calculated from comparison to the Fc control group. The results are shown in Table 32.

TABLE 32 Relative Tumor % Decrease in Antibody Perfusion Tumor Perfusion Fc protein 8.09 ± 2.16 — H1H685P 6.32 ± 2.81 22 VEGF Trap 6.99 ± 1.36 14 H1H685P + 2.46 ± 0.34 70 VEGF Trap

Consistent with the enhanced effect of the combination treatment on perfusion, anti-CD31 staining of tumor sections demonstrated a more potent effect of the combination on tumor blood vessel density (data not shown). The increased effect of the H1H685P+VEGF Trap combination on the function of the tumor vasculature provides a potential explanation for the enhanced effects of the combination therapy on tumor growth.

Example 11. Inhibition of Tumor Growth by a Combination of an Anti-Ang-2 Antibody and a Chemotherapeutic Agent

To test the effect of H1H685P in combination with a chemotherapeutic agent on tumor growth, 2.5×10⁶ COLO 205 tumor cells were implanted subcutaneously into the right hind flank of 8-9 week-old male SCID mice. When the tumors reached an average volume of ˜150 mm³ (day 17 after implantation), mice were randomized into 4 groups (n=5 mice/group) and treated as follows: the first group was treated sc with 15 mg/kg hFc and intraperitoneally (ip) with 5-FU vehicle; the second group was treated sc with 15 mg/kg of H1H685P; the third group was treated ip with 75 mg/kg of 5-FU; the fourth group was treated with the combination of 15 mg/kg H1H685P sc plus 75 mg/kg 5-FU ip. Mice received a total of three treatments, administered every 3-4 days. Tumor volumes were measured twice per week over the course of the experiment. Average (mean+/−standard deviation) tumor growth from the beginning of treatment until day 38 was calculated for each group. Percent decrease of tumor growth was calculated from comparison to the control group. The results are shown in Table 33.

TABLE 33 Avg Tumor Growth % Decrease in Treatment (mm³) Tumor Growth Fc protein + 5-FU  574 ± 110 — vehicle H1H685P 405 ± 80 29 5-FU 313 ± 60 45 H1H685P + 5-FU 175 ± 78 70

The results of this experiment show that the combination of H1H685P and 5-FU caused a greater decrease in tumor growth than either agent administered separately.

Example 12. Anti-Ang-2 Antibodies Attenuate Ocular Angiogenesis In Vivo

In this Example, the effects of selected anti-Ang-2 antibodies on retinal vascularization in a mouse model was assessed.

In one set of experiments wild-type mice were used. In another set, mice expressing a human Ang-2 in place of the wild-type mouse Ang-2 (designated “hu-Ang-2 mice”) were used. The mice at two days of age (P2) were injected subcutaneously with either control Fc or with selected anti-Ang-2 antibodies at a dose of 12.5 mg/kg. Three days later (at P5), pups were euthanized, and eyeballs were enucleated and fixed in 4% PFA for 30 minutes. Retinas were dissected, stained with Griffonia simplicifolia lectin-1 for 3 hours or overnight at 4° C. to visualize the vasculature, and flat-mounted on microscope slides. Images were taken using a Nikon Eclipse 80i microscope camera and analyzed using Adobe Photoshop CS3, Fovea 4.0, and Scion 1.63 software.

Areas of the retina covered with superficial vasculature were measured and used as a readout of antibody activity. The reduction in the size of the vascular areas in mice treated with antibody compared to Fc-treated controls is presented in Table 34. The percent reduction in vascular area reflects the anti-angiogenic potency of the antibody. (N/D=not determined)

TABLE 34 % Reduction in Vascular Area Relative to Fc Control Antibody Wild-Type Mice hAng-2 Mice H1H685P 39.7 N/D H1H690P 30.7 41.5 H1H691P 30.4 N/D H1H696P 31.1 N/D H1H724N 32.2 33.2 H1H744N 35.8 50.5 Control I (Ab) 26.9 35.6

As shown in this Example, the selected anti-Ang-2 antibodies of the present invention substantially inhibited ocular angiogenesis in vivo, thus reflecting the likely anti-angiogenic potential of these antibodies in other therapeutic contexts.

Example 13. Amino Acids of Ang-2 Important for Antibody Binding

To further characterize binding between hAng2 and anti-hAng2 mAbs of the invention, seven variant hAng2-FD-mFc proteins were generated, each containing a single point mutation. Amino acids selected for mutation were based on the difference in sequence between hAng-2 and hAng-1 in the region that interacts with hTie-2 (FIG. 1). In particular, amino acids within the fibrinogen-like domain (FD) of Ang-2 which are believed to interact with Tie-2 based on crystal structure analysis, but which differ from the corresponding amino acid in Ang-1, were individually mutated to the corresponding hAng-1 residue. The results of this example indicate the amino acid residues of hAng-2 with which the Ang-2 preferential binding antibodies interact. That is, if a particular residue (or residues) of hAng-2 is/are changed to the corresponding residue of hAng-1, and the binding of an Ang-2 preferential binding antibody is substantially reduced, then it can be concluded that the antibody interacts with that particular residue(s) of hAng-2.

In this experiment, each of the seven hAng-2FD-mFc mutant proteins were captured (˜147-283 RU) on an anti-mouse-Fc surface created through direct chemical coupling to a BIACORE™ chip. Then each Ang-2 antibody (or peptibody, as the case may be) at 100 nM was injected over the captured mFc-tagged hAng-2FD protein surface at a flowrate of 50 μl/min for 180 sec, and the dissociation of variant hAng2-FD-mFc and antibody was monitored in real time for 20 min at 25° C. Results are summarized in Tables 35a-35d and FIG. 3.

TABLE 35a Mutated hAng-2 H1H685P H1H744N Amino Acid(s)^([1]) RU K_(D) (M) T ½ (min) RU K_(D) (M) T ½ (min) WT^([2]) 210.70 2.23E−11 1988 213 3.98E−11 904 S-417-I 127.65 3.05E−11 1809 127 5.12E−11 1590 K-432-N 152.68 1.40E−11 4468 137 4.87E−11 1690 I-434-M 235.95 1.79E−11 3600 213 3.18E−11 2589 N-467-G 152.25 9.38E−12 6762 139 7.72E−11 1011 F-469-L 101.16 1.38E−08 4 180 1.95E−10 237 Y-475-H 181.53 1.96E−10 289 247 3.06E−10 136 S-480-P 161.13 2.05E−10 289 228 2.25E−11 2129

TABLE 35b Mutated hAng-2 Control I (Ab) Control II (Pb) Amino Acid(s)^([1]) RU K_(D) (M) T ½ (min) RU K_(D) (M) T ½ (min) WT^([2]) 195.25 4.69E−10 54.33 67.44 4.29E−10 39.86 S-417-I 142.96 5.79E−10 32.81 49.99 1.88E−10 36.38 K-432-N 189.69 3.49E−10 51.75 63.21 1.39E−10 42.34 I-434-M 282.10 4.64E−10 48.80 89.15 1.36E−10 57.09 N-467-G 180.90 4.61E−10 44.66 60.94 1.54E−10 46.97 F-469-L 173.01 1.05E−09 25.13 46.73 2.40E−10 36.20 Y-475-H 170.05 1.15E−08 1.85 74.79 1.40E−10 54.12 S-480-P 181.32 2.98E−09 13.36 71.90 1.79E−10 45.45

TABLE 35c Mutated hAng-2 Control III (Pb) Control V (Ab) Amino Acid(s)^([1]) RU K_(D) (M) T ½ (min) RU K_(D) (M) T ½ (min) WT^([2]) 80.33 2.07E−11 170.03 214.48 7.97E−10 48.43 S-417-I 57.13 5.31E−11 114.81 126.45 2.40E−09 29.17 K-432-N 79.22 2.88E−11 200.94 149.14 8.48E−10 75.59 I-434-M 116.22 2.15E−10 62.77 214.75 2.23E−09 31.76 N-467-G 74.64 8.90E−11 109.07 146.77 1.11E−09 55.66 F-469-L 72.66 2.74E−10 66.11 131.96 1.37E−08 1.46 Y-475-H 76.21 6.87E−09 4.11 260.93 2.66E−10 93.22 S-480-P 77.93 2.78E−09 11.69 177.10 3.47E−09 10.33

TABLE 35d Negative Control Mutated hAng-2 (irrelevant antibody) Amino Acid(s)^([1]) RU K_(D) (M) T½ (min) WT^([2]) 0.81 N/B N/B S-417-I −1.21 N/B N/B K-432-N −0.38 N/B N/B I-434-M −1.31 N/B N/B N-467-G −1.09 N/B N/B F-469-L 0.32 N/B N/B Y-475-H −0.20 N/B N/B S-480-P −0.52 N/B N/B ^([1])Amino acid numbering is based on the amino acid numbering of SEQ ID NO: 518. ^([2])WT = wild-type Ang-2FD-mFc construct. N/B = no binding observed.

For purposes of the present invention, an anti-Ang-2 antibody is deemed to interact with a particular Ang-2 amino acid residue if, when the residue is mutated to the corresponding residue of Ang-1, the T½ of dissociation is at least 5-fold less than the T½ of dissociation observed for the wild-type construct under the experimental conditions used in this Example. In view of this definition, antibody H1H685P appears to be unique among the antibodies tested in that it interacts with F469, Y475 and S480. Since H1H685P is also unique because of its strong preferential binding to Ang-2 over Ang-1, it can be concluded that F469, Y475 and S480 comprise an epitope that enables the immunological distinction of Ang-2 from Ang-1. The other antibodies/peptibodies tested in this experiment appear to interact with at most one or two of these residues; i.e., H1H744N and Control I interact with Y475; Control III interacts with Y475 and S480; and Control V interacts with F469. Interestingly, Control II, which was shown to block both Ang-1 and Ang-2 binding to Tie-2 with equal potency, does not interact with any of the Ang-2-specific amino acids identified in this experiment.

Example 14. Ascending, Multiple-Dose Clinical Trial of Intravenously Administered Anti-Ang-2 Antibody (mAb1) in Patients with Advanced Cancer

A. Study Design

This study was an open-label, ascending, multiple-dose study of mAb1 administered as an intravenous (IV) infusion (30 minutes) at doses of 1, 3, 6, 12 or 20 mg/kg once every 2 weeks. The study was conducted in patients with advanced solid tumor malignancies who have failed conventional therapy. The purpose of the study was to explore the safety, recommended dose (RD), pharmacokinetics (PK), pharmacodynamics (PD) and anti-tumor activity of mAb1 (also known as “H1H685P”).

Patients were screened to determine eligibility within 28 days prior to the first dose administration. Three patients were assigned to each of the five sequential cohorts (1, 3, 6, 12 or 20 mg/kg). Patients received mAb1 as a 30 minute IV infusion on Days 1 and 15 of each 4-week cycle (28 days). The dose of mAb1 was escalated until the recommended dose was determined, based on safety, tolerability, PK, PD testing and/or maximum tolerated dose (MTD). The MTD is defined as the highest dose at which less than 33% of patients experience dose-limiting toxicity (DLT) during the first cycle of therapy.

Dose Escalation

Six dose levels were planned for studying dose escalation. Starting at dose 1, a minimum of 3 patients was entered at each dose level. If none of the three patients exhibited a DLT during cycle 1, dose escalation proceeded to the next higher dose level. If one of 3 patients exhibited DLT during cycle 1, the cohort was expanded to 6 patients. If no further cycle 1 DLT was observed, dose escalation proceeded to the next dose level. If two or more patients experienced a cycle 1 DLT, dose escalation was stopped and the previous lower dose level was expanded to 6 patients. Once the RD was determined, up to 10 additional patients were enrolled to evaluate safety and other biological/clinical effects. A separate cohort was planned for patients with hepatocellular carcinoma (HCC) where up to 10 patients were enrolled based on the eligibility criteria (listed below).

A DLT is defined by any of the following treatment-related events occurring during the first cycle (28 days) of study treatment: (1) Hematologic toxicity as evidenced by (a) Grade 3 or 4 neutropenia complicated by fever 38.5° C. or infection; (b) Grade 4 neutropenia of at least 7 days duration; (c) Grade 3 thrombocytopenia complicated by hemorrhage; or (d) Grade 4 thrombocytopenia; and/or (2) Non-hematologic toxicity as evidenced by (a) any Grade 4 non-hematologic toxicity; or (b) Grade 3 non-hematologic toxicities except fatigue, anorexia, nausea, vomiting or diarrhea that can be controlled by medication. Grade 3 laboratory changes that reflected tumor burden were not considered as DLT.

The inclusion criteria for patients were: (1) Male of female of age ≥18 years; (2) histologically or cytologically confirmed diagnosis of malignancy; (3) failure of conventional therapy; (4) ECOG performance status 0-1; (5) resolution of toxicity from prior therapy to Grade ≤2; (6) albumin >35 g/L; (7) normal lactate dehydrogenase (LDH) level; (8) hepatic function as characterized by a) total bilirubin ≤1.5× upper limit of normal (ULN) (if liver metastases ≤3×ULN), and/or b) alanine aminotransferase (ALT) ≤2.5×ULN (or ≤5×ULN, if liver metastases); (9) renal function as characterized by calculated creatinine clearance >60 mL/min; (10) bone marrow function as characterized by a) hemoglobin >9.0 g/dL, b) absolute neutrophil count (ANC) >1.5×10⁹/L, and/or c) platelet count >100×10⁹/L; (11) International normalized ratio (INR)/partial thromboplastin time (PTT) ≤1.5×ULN; (12) for women of childbearing potential, a negative pregnancy test (serum or urine); (13) for men and women of childbearing potential, willingness to use adequate contraception during the course of the study; (14) signed informed consent form and ability to comply with scheduled visits, treatment plans, lab tests and other study procedures.

The exclusion criteria for the study were: (1) brain metastases, spinal cord compression, carcinomatous meningitis or other evidence of central nervous system CNS) involvement; (2) surgery ≤4 weeks prior to the initial administration of mAb1; (3) incomplete wound healing; (4) investigational treatment <4 half-lives prior to the initial administration of mAb1; (5) prior treatment with angiopoietin-1/2 or Tie-2 inhibitors; (6) bleeding diathesis or active bleeding; (7) diastolic BP >90 mmHg; (8) systolic BP >150 mmHg; (9) any of the following ≤6 months prior to the initial administration of mAb1: a) myocardial infarction, unstable angina pectoris, coronary/peripheral artery bypass graft, congestive heart failure (New York Heart Association (NYHA) class III or IV), or ventricular arrhythmia, b) cerebrovascular accident, transient ischemic attack, or Grade ≥2 peripheral neuropathy, and/or c) peptic ulcer disease, erosive esophagitis, or gastritis, infectious or inflammatory bowel disease, or diverticulitis; (10) history of deep vein thrombosis or pulmonary embolism; (11) breast-feeding or pregnancy; and (12) any acute or chronic medical or psychiatric condition or lab abnormality that, in the judgment of the investigator, made the patient inappropriate for entry into the study.

The inclusion criteria for the HCC cohort were: (1) Male or female aged >18 years; (2) Diagnosis of HCC by histopathology or radiologic confirmation of advanced HCC. Hepatocellular carcinoma can be diagnosed radiologically, without the need for biopsy if the typical imaging features are present per American Association for the Study of Liver Diseases (AASLD 2010); (3) ECOG performance status 0-1; (4) Resolution of toxicity from prior therapy (except alopecia) to grade <2; (5) Total bilirubin ≤3×ULN; (6) Albumin ≥3 g/dL; (7) ALT ≤5.0×ULN; (8) Aspartate aminotransferase (AST) ≤5.0×ULN; (9) Creatinine <1.5×ULN; (10) Spot urine protein <2+ (dipstick method) or proteinuria <1 g/24 hr; (11) Hemoglobin ≥9.0 g/dL; (12) Absolute neutrophil count (ANC) ≥1.5×10⁹/L; (13) Platelet count ≥75×10⁹/L; (14) Adequate coagulation function by INR ≤2; (15) At least 3 weeks must have elapsed since the last dose of chemotherapy, hormonal therapy (luteinizing hormone releasing hormone [LHRH] agonist is allowed for patients with castrate resistant prostate cancer), or radiotherapy (6 weeks for nitrosoureas, mitomycin C, or cytokine therapy) prior to the initial administration of mAb1 (Note: the 3-week waiting period for radiotherapy does not apply to localized radiation therapy for pain control); (16) For patients who have received prior local therapy (RFA or TACE), at least 4 weeks must have elapsed between receipt of the prior treatment and the first dose of mAb1; there must be evidence of tumor growth or untreated evaluable tumor; and toxicity related to the prior therapy must have returned to baseline, resolved, or be considered irreversible; (17) At least 6 weeks must have elapsed since the last dose of bevacizumab (Avastin™) prior to the initial administration of mAb1; (18) At least 3 weeks must have elapsed since the last dose of sorafenib prior to the initial administration of mAb1; (19) At least 4 weeks must have elapsed since the last surgery prior to the initial administration of mAb1; (20) At least 4 weeks must have elapsed since the last dose of investigational treatment prior to the initial administration of mAb1; (21) For women of childbearing potential, a negative pregnancy test (serum) within 72 hours prior to the initial administration of mAb1; (22) For women of childbearing potential and men whose sexual partners are of childbearing potential, willingness to use adequate contraception during the course of the study and for 30 days after last dose of mAb1; (23) Ability and willingness to comply with scheduled visits, treatment plans, laboratory tests, and other study procedures; and (24) Signed informed consent form (ICF).

The exclusion criteria for the HCC cohort were: (1) Brain metastases, spinal cord compression, carcinomatous meningitis, or other evidence of central nervous system involvement; (2) Decompensated cirrhosis with clinical, significant or intractable ascites and/or encephalopathy); (3) Serious non healing wound or acute ulcer; (4) Prior treatment with Ang2 or Tie-2 inhibitors; (5) Bleeding diathesis or active bleeding (bleeding from varices, treatment for bleeding from varices, or at risk for varices bleeding); (6) Anticoagulation therapy, except aspirin at doses of 325 mg or less per day or low-dose warfarin; (7) Abnormal 12-lead ECG of clinical significance; (8) Either systolic blood pressure >150 mm Hg or diastolic blood pressure >95 mm Hg on at least 2 repeated determinations on separate days within the screening period. Patients who were excluded by these criteria were rescreened after adequate control and stabilization of their blood pressure on repeat measurements; (9) Any of the following ≤6 months prior to the initial administration of mAb1: a) Myocardial infarction, unstable angina pectoris, coronary/peripheral artery bypass graft, congestive heart failure (NYHA class III or IV) or ventricular arrhythmia, and/or b) Cerebrovascular accident or transient ischemic attack; (10) Grade >2 peripheral neuropathy; (11) Deep vein thrombosis or pulmonary embolism within last 6 months prior to initial administration of mAb1. Asymptomatic portal vein thrombosis allowed; (12) Breast-feeding or pregnancy; (13) Any acute or chronic medical or psychiatric condition that, in the judgment of the investigator, made the patient inappropriate for entry into the study; and (14) Hypersensitivity to any compound in the tetracycline antibiotics group.

B. Study Objectives

The primary objectives of the study were: (1) to study the safety, tolerability and DLTs of IV administered mAb1; (2) to determine the RD of mAb1 administered IV every 2 weeks in patients with advanced solid malignancies; and (3) to assess the safety of mAb1 administered at the RD every 2 weeks to patients with advanced HCC or other advanced solid malignancies.

The secondary objectives of the study were: (1) to characterize the PK profile of mAb1; (2) to assess the immunogenicity of mAb1; (3) to study the preliminary anti-tumor activity of mAb1 administered to patients with HCC and other advanced solid malignancies; and (4) to evaluate correlative biomarkers of mAb1 activity.

C. Treatment

The study drug mAb1 was supplied as a liquid in sterile, single-use vials. Each vial contained a withdrawable volume of 10 mL of mAb1 at a concentration of 25 mg/mL. mAb1 was administered in an outpatient setting. Each patient's dose depended on individual body weight and assigned dosing cohort. The dose was adjusted each cycle for variations in body weight in kilograms if weight changed by ≥10%. mAb1 was administered once every 2 weeks as a 30-minute IV infusion. A cycle was defined as 28 days.

For IV administration, mAb1 was diluted into an IV bag of 0.9% sodium chloride injection US Pharmacopeia under aseptic conditions. Dilution of mAb1 into an IV bag of normal saline to concentrations below 1.0 mg/mL of mAb1 required the addition of diluent to stabilize the drug during delivery through the IV infusion apparatus. The infusion sets included a 0.2 μm polyethersulfone inline filter.

D. Statistical Analysis

The analysis of the study was descriptive and exploratory in nature. Results were summarized by cohort. A 3+3 dose escalation rule was used. Data was summarized using descriptive statistics only (number of patients, mean, median, standard deviation, minimum and maximum) for continuous variables and using frequency and percentages for discrete variables. Safety observations and measurements including drug exposure, adverse events, lab data, vital signs and ECOG performance status was summarized.

E. Safety Assessment

Safety was assessed throughout the study by monitoring Adverse Events and Serious Adverse Events.

An Adverse Event (AE) is any untoward medical occurrence in a subject or clinical investigation subject administered a pharmaceutical product. An AE can, therefore, be any unfavorable and unintended sign (including abnormal laboratory finding), symptom, or disease temporally associated with the use of a medicinal product, whether or not considered related to the medicinal (investigational) product. AEs also include: any worsening (i.e., any clinically significant change in frequency and/or intensity) of a pre-existing condition that is temporally associated with the use of the study drug; abnormal laboratory findings considered by the Investigator to be clinically significant; and any untoward medical occurrence.

A Serious Adverse Event (SAE) is any untoward medical occurrence that at any dose results in death; is life-threatening; requires in-patient hospitalization or prolongation of existing hospitalization; results in persistent or significant disability/incapacity; is a congenital anomaly/birth defect; or is an important medical event.

Safety assessments carried out at screening/baseline and during the study include physical examination (including complete neurological assessment at onset of symptoms or upon any brain MRI abnormal findings in any patient), laboratory tests, vital signs, brain MRI, pregnancy test, and tumor assessment.

The clinical laboratory data consists of hematology, blood chemistry and urinalysis. Blood samples for hematology testing were collected at every study visit; blood samples for serum chemistry testing and urine samples for urinalysis were collected to measure overall patient health at screening, day 1/baseline (pre-dose), day 8, day 15, and day 22 of the first cycle, and days 1, and 15 of subsequent cycles, or early termination if subject is discontinued from the study.

Vital sign parameters include respiratory rate (bpm), pulse rate (bpm), systolic and diastolic blood pressure (mmHg) and body temperature (° C.). Vital signs were collected (pre-dose, on dosing days) at screening and day 1/baseline, and days 8, 15, and 22 of the first cycle, and days 1, and 15 of subsequent cycles, or early termination. Vital signs were taken at 1 and 2 hours post-infusion following the study drug dose on days 1, and 15.

12-Lead ECG parameters include: Ventricular HR, PR interval, QRS interval, corrected QT interval (QTcF=QT/[RR^(0.33)] and QTcB=QT/[RR^(0.5)]) ECG status: normal, abnormal not clinical significant or abnormal clinical significant. A standard 12-lead ECG was performed at screening, and 30 days-post last dose (end of study) or early termination.

A thorough and complete physical examination was performed at screening, days 1 and 15 of each cycle, and 30 days-post last dose (end of study) or early termination.

A brain MRI was performed during screening (within 28 days prior to start of study drug administration). A brain MRI was also performed during even numbered cycles between days 15 and 28 and at the 30 day follow-up.

For tumor assessment, a CT or MRI was performed at the screening visit (within 28 days prior to infusion) and during every even-numbered cycle (eg cycles 2, 4, 6, 8) between days 15 and 28 and when disease progression was suspected. Tumor assessment was performed according to Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST version 1.1). RECIST criteria are developed to facilitate objective assessment of change in tumor burden such as tumor shrinkage and time to development of disease progression. RECIST guidelines are posted on the worldwide web at http://ctep(dot)cancer(dot)gov(slash)protocoldevelopment(slash)docs(slash)recist_guideline(dot)pdf. Tumor marker assessments were also collected, when available.

In addition, tumor and skin biopsies were obtained once before and once after treatment: at baseline and in cycle 1, on day 22.

Patients in the HCC cohort were additionally screened for hepatitis-B, hepatitis-C and for alpha-fetoprotein. Dynamic contrast-enhanced ultrasound imaging was performed before the tumor biopsy during screening and on day 22 of cycle 1.

F. Pharmacokinetic Studies

Blood samples were collected for RNA, serum cytokine testing, molecular profiling, anti-drug antibody (ADA) and serum mAb1 levels at screening and post-infusion on days 1, 2, 3, 8, 15 and 22 of cycle 1, and on day 1 of subsequent cycles. Tumor biopsies were obtained for immunohistochemistry studies for Ang-2. Results were scored as positive for Ang-2 in association with expression of endothelial cell CD31.

G. Preliminary Results

37 patients [17M/20F; median age 43.5 (range 22-82); ECOG PS 0(9)/1(28)] were enrolled. Twenty-three (23) patients were enrolled in the 1 (4), 3 (4), 6 (3), 12 (6) and 20 mg/kg (6) dose escalation cohorts. No DLTs were reported and a MTD was not reached. Fourteen (14) patients were enrolled in a 12 mg/kg expansion cohort; enrollment at 20 mg/kg expansion was started. Most common G½ treatment-related adverse events (TRAEs) were fatigue (19%), peripheral edema (17%), diarrhea (14%), abdominal distension (11%), and decreased appetite (11%). There were no ≥Grade 3 TRAEs. A confirmed sustained patient response (PR) (16 wks) was observed in 1 patient with adrenocortical cancer treated at 1 mg/kg. Stable disease (SD) (range 3.3-46.8 weeks) was reported for 17 of 32 (53%) patients evaluable for efficacy. A total of 16 patients received treatment >16 weeks. One patient with thyroid cancer had SD for 46 weeks. Across all dose levels, mAb1 pharmacokinetics (PK) appeared linear and dose-proportional. The PK profile is characterized by an initial distribution and a single mono-exponential elimination phase. In 1 patient (3 mg/kg), positive ADA was detected. Total circulating serum Ang2 levels appeared saturated following treatment in all cohorts, indicating systemic target engagement at all doses tested.

H. Conclusions

Administration of mAb1 in patients with advanced cancer was generally well tolerated, with mainly mild and moderate TRAEs. Dose escalation was completed, and enrollment to the 20 mg/kg expansion continued. The safety profile supported combination with chemotherapy or other anti-angiogenic agents.

Example 15. Clinical Trial of Combined Administration of Anti-Angiogenesis Anti-Ang-2 Antibody and Aflibercept in Patients with Advanced Solid Tumor Malignancies

A. Study Design

The study consists of 2 phases. Phase 1b is an open-label, multi-centered, dose-escalation study of mAb1 (also known as “H1H685P”) in combination with aflibercept (also known as “VEGF Trap”; see U.S. Pat. No. 7,087,411) administered in patients with advanced platinum-resistant ovarian cancer (OC), colorectal cancer (CRC) or HCC. Four dose levels of mAb1/aflibercept will be studied in a standard “3+3” design (up to 6 patients per cohort): mAb1 (3, 6 and 12 mg/kg) combined with a fixed dose of aflibercept (2 mg/kg). An additional cohort will then be enrolled using the highest tolerable dose combination of mAb1 in combination with 4 mg/kg of aflibercept. A total of 6 patients will be dosed at the highest dose combination of mAb1 and aflibercept prior to declaring the recommended dose (RD) for the second phase of the study.

Phase 2 is a double-blind, followed by an open-label, multi-centered, randomized study in patients with advanced platinum-resistant OC. Three arms (each with n=46) will be studied:

Arm 1: mAb1+aflibercept until PD (determined by RECIST 1.1)

Arm 2: mAb1 until PD (determined by RECIST 1.1), followed by mAb1+aflibercept until PD on the combination treatment (determined by RECIST 1.1, and using the measurements upon the switch as a new baseline)

Arm 3: Aflibercept until PD (determined by RECIST 1.1) followed by mAb1+aflibercept until PD on the combination treatment (determined by RECIST 1.1, and using the measurements upon the switch as a new baseline).

Patients will be screened to determine eligibility within 28 days prior to the initial administration of mAb1 and aflibercept. All patients will be observed for the occurrence of a dose-limiting toxicity (DLT) during cycle 1. The maximum tolerated dose (MTD) is defined as the highest dose level at which no more than 1 DLT out of 6 patients has been documented. The recommended Phase 2 dose (RP2D) will be determined based on a review of the safety (all cycles), PK, PD and efficacy observations documented during the dose escalation. The RP2D may be the same or lower than the MTD.

The study drug will be administered every 2 weeks. For Phase 1b, mAb1 will be administered as a 30-minute IV infusion, followed by aflibercept as a 60-minute IV infusion for a total infusion time of 90 minutes. The study drug will be administered on cycle days 1 and 15 of each 28-day cycle. Treatment will continue till disease progression, unacceptable toxicity or withdrawal of consent.

For Phase 2, the administration of the study drug for each of the three arms will be as follows:

Arm 1: Double blind administration of mAb1 as a 30-minute IV infusion, followed by aflibercept as a 60-minute IV infusion for a total infusion time of 90 minutes every 2 weeks until disease progression.

Arm 2: Double-blind administration of mAb1 as a 30-minute IV infusion, followed by normal saline as a 60-minute IV infusion for a total infusion time of 90 minutes every 2 weeks until disease progression. Then, open-label administration of mAb1 as a 30-minute IV infusion, followed by aflibercept as a 60-minute IV infusion for a total infusion time of 90 minutes every 2 weeks until disease progression. Arm 3: Double blind administration of normal saline as a 30-minute IV infusion, followed by aflibercept as a 60-minute IV infusion for a total infusion time of 90 minutes every 2 weeks until disease progression. Then, open label administration of mAb1 as a 30-minute IV infusion, followed by aflibercept as a 60-minute IV infusion for a total infusion time of 90 minutes every two weeks until disease progression.

The study drug will be administered on cycle days 1 and 15 of each 28-day cycle. The dose is defined by the RP2D Phase 1b. Treatment will continue in each arm till disease progression, unacceptable toxicity or withdrawal of consent.

The patient inclusion criteria for Phase 1b are: (1) male or female of age ≥18 years; (2) confirmed diagnosis of one of the following—a) epithelial adenocarcinoma of the ovary (including fallopian tube or primary peritoneal cancer) by histology, b) unrespectable or metastatic colorectal adenocarcinoma by histology or cytology, or c) advanced hepatocellular cancer by histopathology or radiologic confirmation. HCC can be diagnosed radiologically, without the need for biopsy if the typical imaging features are present per American Association for the Study of Liver Diseases (AASLD 2010); (3) no standard therapeutic options of proven benefit; (4) ECOG performance status 0 or 1; (5) bone marrow function as characterized by a) hemoglobin >9.0 g/dL, b) absolute neutrophil count (ANC) >1.5×10⁹/L, and/or c) platelet count >75×10⁹/L; (6) hepatic function as characterized by a) total bilirubin ≤1.5×ULN (≤3×ULN, if liver metastases or HCC), b) transaminases ≤2.5×ULN (or ≤5.0×ULN, if liver metastases or HCC), and/or c) alkaline phosphatase ≤2.5×ULN (or ≤5.0×ULN, if liver metastases or HCC); (7) renal function as characterized by a) serum creatinine ≤1.5 mg/dL, and/or b) spot urine protein <2+ (dipstick method) or proteinuria <1 g/24 hr; (8) calculated creatinine clearance >60 mL/min (Cockroft-Gault formula); (9) albumin >3.5 g/dL; (10) resolution of toxicity from prior therapy to Grade ≤1, including skin toxicity; (11) negative serum pregnancy test in women of child-bearing potential; (12) willingness to use adequate forms of contraception for males and females of child-bearing potential or avoidance of intercourse during the study; (13) patients capable of understanding and signing an informed consent form; and (14) ability to comply with scheduled visits, treatment plans, lab tests and other study procedures.

The exclusion criteria for Phase 1b are: (1) any active or previously treated brain metastases, spinal cord compression, carcinomatous meningitis, or other evidence of CNS involvement; (2) appearance in brain MRI at screening of any lesion that demonstrates contrast enhancement with T1 weighted imaging; (3) abnormal 12-lead ECG of clinical significance; (4) either systolic BP >150 mmHg or diastolic BP >95 mmHg on at least 2 repeated determinations on separate days within the screening period; (5) any of the following ≤6 months prior to the initial administration of mAb1/aflibercept—a) myocardial infarction, unstable angina pectoris, coronary/peripheral artery bypass graft, congestive heart failure or ventricular arrhythmia, b) cerebrovascular accident or transient ischemic attack, c) deep vein thrombosis or pulmonary embolism (including asymptomatic pulmonary embolism identified on imaging), d) serious non-healing wound or acute ulcer, and/or e) bleeding diathesis or active bleeding (bleeding from varices, treatment for bleeding from varices, or at risk for varices bleeding); (6) anticoagulation therapy, except aspirin at doses of 325 mg or less per day or low-dose warfarin or heparin; (7) major surgery <4 weeks prior to initial administration of mAb1/aflibercept; (8) IV port placement <7 days prior to initial administration of mAb1/aflibercept; (9) bleeding peptic ulcer disease, erosive gastritis, intestinal perforation or clinically significant GI hemorrhage within 6 months of enrollment; (10) transmural tumor bowel involvement; (11) history of abdominal or tracheal-esophageal fistula; (12) for HCC patients—decompensated cirrhosis with clinical, significant or intractable ascites and/or encephalopathy; (13) prior treatment with Ang2 or Tie-2 inhibitors; (14) prior treatment with aflibercept; (15) bevacizumab administration within 6 weeks of first study drug administration; (16) investigational treatment <5 half-lives prior to the initial administration of mAb1/aflibercept; (17) history of allergic reactions or acute hypersensitivity reaction attributable to antibiotics of the tetracycline class; (19) concurrent severe medical problems that would significantly limit patient compliance with the study requirements or interfere with the interpretation of study results; (20) breast-feeding or pregnancy; and (21) acute or chronic psychiatric problems that, under the evaluation of the investigator, make the patient ineligible for participation.

The inclusion criteria for Phase 2 are: (1) female of age ≥18 years; (2) confirmed diagnosis of epithelial adenocarcinoma of the ovary (including fallopian tube or primary peritoneal cancer) by histology; (3) platinum-resistant or refractory disease (disease recurrence within 6 months of last dose of platinum or disease progression while treated with platinum-based regimen); (4) measurable disease; (5) ECOG performance status 0 or 1; (6) at least 2 pretreatment samples minimum 2 weeks apart indicating CA125≥2×ULN; (7) bone marrow function as characterized by—a) hemoglobin >9.0 g/dL, b) ANC >1.5×10⁹/L, and/or c) platelet count >75×10⁹/L; (8) hepatic function as characterized by a) total bilirubin ≤1.5×ULN (≤3×ULN, if liver metastases or HCC), b) transaminases ≤2.5×ULN (or ≤5.0×ULN, if liver metastases or HCC), and/or c) alkaline phosphatase ≤2.5×ULN (or ≤5.0×ULN, if liver metastases or HCC); (9) renal function as characterized by a) serum creatinine ≤1.5 mg/dL, and/or b) spot urine protein <2+ (dipstick method) or proteinuria <1 g/24 hr; (10) calculated creatinine clearance >60 mL/min (Cockroft-Gault formula); (11) albumin >3.5 g/dL; (12) resolution of toxicity from prior therapy to Grade ≤1, including skin toxicity; (13) negative serum pregnancy test in women of child-bearing potential; (14) willingness to use adequate forms of contraception if patient is of child-bearing potential or avoidance of intercourse during the study; (15) patients capable of understanding and signing an informed consent form; and (16) ability to comply with scheduled visits, treatment plans, lab tests and other study procedures.

The exclusion criteria for Phase 2 are the same as given above for Phase 1b, with the addition of 2 more criteria: (1) more than 3 prior lines of platinum-based systemic therapies; and (2) more than 7 prior lines of systemic therapies.

B. Study Objectives

The primary objectives of the study for Phase 1b are: (1) to assess the safety profile of mAb1 administered in combination with aflibercept in patients with advanced platinum-resistant ovarian cancer (OC), colorectal cancer (CRC) or hepatocellular cancer (HCC); and (2) to determine the recommended phase 2 dose (RP2D) of the mAb1/aflibercept combination.

The secondary objectives of the study for Phase 1b are: (1) to characterize the pharmacokinetic (PK) profile of mAb1 when used in combination with aflibercept; (2) to characterize the PK profile of aflibercept when used in combination with mAb1; (3) to assess the immunogenicity of mAb1 when used in combination with aflibercept; (4) to assess the immunogenicity of aflibercept when used in combination with mAb1; (5) to describe the preliminary anti-tumor activity of mAb1/aflibercept combination in patients with advanced platinum-resistant OC, CRC or HCC; and (6) to explore the potential correlative (safety/efficacy) biomarkers of mAb1/aflibercept activity.

The primary objectives of the study for Phase 2 is to compare objective response rate determined by CA-125 based response criteria of mAb1 alone versus mAb1 combined with aflibercept in patients with advanced platinum-resistant OC.

The secondary objectives of the study for Phase 2 are: (1) to assess the safety profile of mAb1 followed by mAb1/aflibercept and aflibercept followed by mAb1/aflibercept; (2) to compare objective response rate determined by CA-125 of mAb1 alone versus aflibercept alone in patients with advanced platinum-resistant OC; (3) to compare objective response rate determined by CA-125 of mAb1 combined with aflibercept versus aflibercept alone in patients with advanced platinum-resistant OC; (4) to assess objective response rate and disease control rate determined by RECIST 1.1 of mAb1 alone, aflibercept alone and mAb1/aflibercept combination in patients with advanced platinum-resistant OC; (5) to assess disease control rate determined by CA-125 of mAb1 alone, aflibercept alone and mAb1/aflibercept combination in patients with advanced platinum-resistant OC; (6) to assess time to progression and progression-free survival determined by RECIST 1.1 of mAb1 alone, aflibercept alone and mAb1/aflibercept combination in patients with advanced platinum-resistant OC; (7) to assess time to progression and progression free survival determined by CA-125 of mAb1 alone, aflibercept alone and mAb1/aflibercept combination in patients with advanced platinum-resistant OC; (8) to assess landmark six month and one year OS rates of mAb1 alone, aflibercept alone and mAb1/aflibercept combination in patients with advanced platinum-resistant OC; (9) to assess safety and efficacy profile for patients who switched from mAb1 alone to combo mAb1/aflibercept and patients who switched from aflibercept alone to combo mAb1/aflibercept; (10) to characterize the PK profile of mAb1 when used in combination with aflibercept; (11) to characterize the PK profile of aflibercept when used in combination with mAb1; (12) to assess the immunogenicity of mAb1 when used in combination with aflibercept; (13) to assess the immunogenicity of aflibercept when used in combination with mAb1; and (14) to explore the potential correlative biomarkers (safety/efficacy) of mAb1/aflibercept activity.

C. Treatment

mAb1 is administered in combination with aflibercept once every 2 weeks. mAb1 is administered as a 30-minute IV infusion followed by aflibercept as a 60-minute IV infusion, for a total infusion time of 90 minutes.

mAb1 is supplied in sterile single use vials at a withdrawable volume of 10 mL at a concentration of 25 mg/mL. For IV administration, mAb1 is diluted into an IV bag of 0.9% sodium chloride injection US Pharmacopeia under aseptic conditions. The infusion sets include a 0.2 μm polyethersulfone inline filter.

Aflibercept is supplied as a sterile, non-pyrogenic, colorless to pale-yellow-colored, 25 mg/mL solution, packaged in a type 1 clear glass vial stopped with an elastomeric closure and sealed with an aluminum seal. Prior to infusion, aflibercept is diluted directly into infusion bags of 0.9% injection US Pharmacopeia or 5% dextrose. The concentration of the diluted solution can range between 0.6 and 8 mg/mL. Dilution must be carried out under aseptic conditions as aflibercept does not contain any microbial preservative. The infusion sets include a 0.2 um polyethersulfone inline filter.

D. Statistical Analyses

The analyses for the initial phase will be descriptive and exploratory in nature. Results for the pre-specified endpoints will be summarized by cohort for each arm. Statistical considerations will be detailed in the statistical analysis plan (SAP).

For the Phase 2, stratification will be included for patients with prior bevacizumab. Based on assumption of a CA-125 response rate of 30% mAb1 alone and 60% response rate for mAb1/aflibercept, 42 patients per arm will have 80% power to detect treatment difference at two-sided 0.05 level by Chi-squared test. With the assumption of cycle 1 dropout rate of 10%, the total number of patients is 138 (46 per arm).

The primary endpoint will be analyzed using Pearson Chi-square test, the 2-sided 95% confidence interval for the difference of two treatment arms will be calculated using normal approximation. The other response type endpoints will be analyzed similarly with primary endpoint. The time-to-events data will be summarized by Kaplan-Meier methods and analyzed by log-rank test, if appropriate. There will be no multiplicity adjustment between the analysis of primary and secondary endpoints in the study.

E. Safety Assessment

Safety will be assessed throughout the study by monitoring Adverse Events and Serious Adverse Events.

An Adverse Event (AE) is any untoward medical occurrence in a subject or clinical investigation subject administered a pharmaceutical product. An AE can, therefore, be any unfavorable and unintended sign (including abnormal laboratory finding), symptom, or disease temporally associated with the use of a medicinal product, whether or not considered related to the medicinal (investigational) product. AEs also include: any worsening (i.e., any clinically significant change in frequency and/or intensity) of a pre-existing condition that is temporally associated with the use of the study drug; abnormal laboratory findings considered by the Investigator to be clinically significant; and any untoward medical occurrence.

A Serious Adverse Event (SAE) is any untoward medical occurrence that at any dose results in death; is life-threatening; requires in-patient hospitalization or prolongation of existing hospitalization; results in persistent or significant disability/incapacity; is a congenital anomaly/birth defect; or is an important medical event.

Safety assessments carried out at screening/baseline and during the study include physical examination (including complete neurological assessment at onset of symptoms or upon any brain MRI abnormal findings in any patient), laboratory tests, vital signs, brain MRI, pregnancy test, and tumor assessment.

The clinical laboratory data consists of hematology, blood chemistry and urinalysis. Blood samples for hematology testing will be collected at every study visit; blood samples for serum chemistry testing and urine samples for urinalysis will be collected to measure overall patient health at screening, day 1/baseline (pre-dose), day 8, day 15, and day 22 of the first cycle, and days 1, and 15 of subsequent cycles, or early termination if subject is discontinued from the study. In addition, other laboratory tests including international normalized ratio/partial thromboplastin test, calculated creatinine clearance test, and hepatitis-B/C will be carried out.

Vital sign parameters include respiratory rate (bpm), pulse rate (bpm), systolic and diastolic blood pressure (mmHg) and body temperature (° C.). Vital signs were collected (pre-dose, on dosing days) at screening and day 1/baseline, and days 8, 15, and 22 of the first cycle, and days 1, and 15 of subsequent cycles, or early termination. Vital signs were taken at 1 and 2 hours post-infusion following the study drug dose on days 1, and 15.

12-Lead ECG parameters include: Ventricular HR, PR interval, QRS interval, corrected QT interval (QTcF=QT/[RR^(0.33)] and QTcB=QT/[RR^(0.5)]) ECG status: normal, abnormal not clinical significant or abnormal clinical significant. A standard 12-lead ECG will be performed at screening, and 30 days-post last dose (end of study) or early termination.

A thorough and complete physical examination will be performed at screening, days 1 and 15 of each cycle, and 30 days-post last dose (end of study) or early termination.

A brain MRI, with and without gadolinium contrast and with T1 and T2 weighted imaging sequences, will be performed during screening (within 28 days prior to start of study drug administration). A brain MRI will also be performed during even numbered cycles between days 15 and 28 and at the 30 day follow-up.

For tumor assessment, a CT or MRI is performed at the screening visit (within 28 days prior to infusion) and during every even-numbered cycle (eg cycles 2, 4, 6, 8) between days 15 and 28 and when disease progression is suspected. Tumor assessment is performed according to Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST version 1.1). RECIST criteria are developed to facilitate objective assessment of change in tumor burden such as tumor shrinkage and time to development of disease progression. RECIST guidelines are posted on the worldwide web at http://ctep(dot)cancer(dot)gov(slash)protocoldevelopment(slash)docs(slash)recist_guideline(dot)pdf. Tumor marker assessments are also collected, when available.

In addition, tumor and skin biopsies will be obtained once before and once after treatment: at baseline and in cycle 1, on day 22.

F. Pharmacokinetic Studies

Blood samples will be collected for RNA, serum cytokine testing, CTAD (citrate, theophylline, adenosine and dipyridamole), molecular profiling, and serum mAb1 and aflibercept levels at screening and post-infusion on days 1, 2, 3, 8, 15 and 22 of cycle 1, on day 1 of subsequent cycles, and on day 30 post-last dose. Results will be used to study drug-related changes in expression of angiogenesis and inflammatory-related genes and proteins (eg, including but not limited to factors such as Ang-2, soluble Tie-2, VEGF, soluble VEGF receptor 2). Tumor biopsies will be obtained for immunohistochemistry studies for Ang-2 to assess the expression of angiogenesis-related factors including, but not limited to, Ang-1/2, VEGF-A/B/C/D, VEGF-R-1/2/3 and placental growth factor. In addition, tumor DNA/RNA will be extracted to determine the expression and potential genetic mutations and/or polymorphisms known to be associated with underlying disease characteristics, tumor progression and/or tumor angiogenesis.

The tumor burden of each patient with platinum-resistant ovarian cancer will be assessed by RECIST 1.1 every 2 cycles (8 weeks) and by serum CA-125 every dosing (2 weeks).

G. Status

The phase 1b study employs a standard 3+3 dose escalation design exploring 5 different combination treatment dose levels of mAb1 and aflibercept. Once the recommended phase 2 dose (RD) of the combination treatment is determined, additional patients will be enrolled in a safety expansion cohort, for a planned total enrollment of up to 40 patients. The primary study objectives are to evaluate the safety and determine the RD of the 2 drugs in combination both administered IV every 2 weeks in patients with advanced solid tumors. Secondary endpoints include characterization of the PK and potential immunogenicity of mAb1 and aflibercept when given in combination, evaluation of correlative PD biomarkers related to mAb1 and aflibercept in addition to exploring preliminary antitumor activity. Enrollment to cohorts 1 and 2 has been completed without DLT.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. 

What is claimed is:
 1. A method for treating advanced hepatocellular cancer, the method comprising: (a) selecting a patient with advanced hepatocellular carcinoma wherein the patient is resistant to conventional anti-tumor therapy; and (b) sequentially administering to the patient in need thereof one or more doses of an antibody or antigen-binding fragment thereof which specifically binds human angiopoietin-2 (hAng-2) but does not substantially bind hAng-1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) from SEQ ID NO: 18, and a light chain variable region (LCVR) comprising three light chain CDRs (LCDR1, LCDR2 and LCDR3) from SEQ ID NO:
 20. 2. The method of claim 1, wherein: (a) the HCDR1 comprises SEQ ID NO: 4; (b) the HCDR2 comprises SEQ ID NO: 6; (c) the HCDR3 comprises SEQ ID NO: 8; (d) the LCDR1 comprises SEQ ID NO: 12; (e) the LCDR2 comprises SEQ ID NO: 14; and (f) the LCDR3 comprises SEQ ID NO:
 16. 3. The method of claim 1, wherein the HCVR comprises SEQ ID NO:
 18. 4. The method of claim 1, wherein the LCVR comprises SEQ ID NO:
 20. 5. The method of claim 1, wherein the HCVR comprises SEQ ID NO: 18 and the LCVR comprises SEQ ID NO:
 20. 6. The method of claim 1, wherein step (b) comprises administering an initial dose comprising the anti-Ang-2 antibody followed by one or more subsequent doses of the anti-Ang-2 antibody.
 7. The method of claim 6, wherein each subsequent dose is administered 2 weeks after the immediately preceding dose.
 8. The method of claim 1, wherein the one or more doses of the anti-Ang-2 antibody comprises 1-20 mg/kg of patient body weight.
 9. The method of claim 1, wherein the antibody or antigen-binding fragment thereof is administered in combination with a vascular endothelial growth factor (VEGF) antagonist.
 10. The method of claim 9, wherein the VEGF antagonist is selected from the group consisting of an anti-VEGF antibody, a small molecule kinase inhibitor of VEGF receptor and a VEGF-inhibiting fusion protein. 